Methods and dressings for sealing internal injuries

ABSTRACT

Disclosed are solid and frozen haemostatic materials and dressings consisting essentially of a fibrinogen component and a fibrinogen activator. Also disclosed are methods of treating internal wounded tissue in a mammal by applying one or more of these haemostatic materials and dressings.

This application is a continuation-in-part application, and thereforeclaims benefit of the filing date, of prior U.S. Provisional PatentApplication No. 60/935,311, the disclosure of which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to dressings for treating internal injuredtissue in a mammalian patient, such as a human and methods of using thesame.

BACKGROUND OF THE INVENTION

There are a large number of medical procedures that result in injuriesto blood vessels. Similarly, there are numerous examples of bleedingcaused by traumatic injuries, hematological disorders, and from unknowncauses. When the site of bleeding is not readily accessible, such as aninjured vessel located deep within the flesh, or inside a body cavity, asimple and effective method of hemorrhage control that can access thesite within the body and seal the injured vessel is needed. Similarly,tissue may be divided by either traumatic injury or surgical procedure,and require sealing to approximate the edges of the injury in order torestore function. Current sealing products and devices have one or moredeficiencies, usually due to their inadequate performance, or theirreliance upon non-natural components that interfere with normal healing.

The need for improved technologies to address these injuries issignificant. For example, in the case of blood vessels that have beendeliberately punctured as part of a diagnostic and/or therapeuticprocedure (such as cardiac catheterization, balloon angioplasty,vascular stenting and the like), over seven million such procedures arecurrently performed every year, but with a 9% overall complication rateand a 1-3% major complication rate (See Millennium Research Group:Global Markets for Vascular Closure Devices 2006). These complicationscan lead to significant morbidity, increased expense, a requirement foradditional procedures and/or devices, extended time in the medicalfacility and conversion of outpatients to inpatients. Commerciallyavailable products now available only reduce the major complication rateby one half of one percent (See Arora et al: Am Heart J. 2007 April;153(4):606-11) to 2.4%. Nevertheless, despite this poor performance,even these devices are currently used since the costs and consequencesof procedure-induced complications is so high (See Resnic et al: Am J.Cardiol. 2007 Mar. 15; 99(6):766-70).

Not only are there the above described complications associated withtherapy itself, closure of the access hole(s) created in the bloodvessel is a significant source of additional complications, includinguncontrolled hemorrhage, pseudoaneurysm, hematoma, arteriovenousfistula, arterial thrombosis, infection, and retained devices (SeeMeyerson et al: Angiographic Access Site Complications in the Era ofArterial Closure Devices Vasc Endovasc Surg, 2002; 36 (2) 137-44). Theseadditional complications may lead to prolonged closure procedures,hospitalization, the requirement for surgical repair, and even tissueloss or death.

Currently, the primary means of closing the access hole in the vesselhas been to allow a natural blood clot to form at the puncture site.This has generally been accomplished by manual compression, but variousproducts have recently been developed in an attempt to reduce the timerequired to achieve vascular closure. Such devices automate theapplication of pressure over the injury site, suture the hole in thevessel, clip the hole shut, or apply some sort of patch or pad thatallegedly increases the formation of a natural clot at the site. Thesedevices are convenient and gaining in popularity, but their overallsafety appears over estimated. Indeed, far from being risk free, thesedevices may be associated with unique levels of hemorrhagic and cardiacrisks including myocardial infarction, stroke and death (See Rao, S.Implications of bleeding and blood transfusion in percutaneous coronaryintervention. Rev Cardiovasc Med. 2007, 8 Suppl 3:S18-26.).

Significant risks have been reported to be associated with all classesof vascular closure devices. Most seriously, the severity and thedifficulty in treating complications are generally greater when vascularclosure devices are used (See Nehler et al. Iatrogenic vascular injuriesfrom percutaneous vascular suturing devices. J. Vasc Surg 2001 May;33(5):943-7; Castelli et al: Incidence of vascular injuries after use ofthe Angio-Seal closure device following endovascular procedures in asingle center. World J. Surg. 2006 March; 30(3):280-4.). The use of suchdevices is even associated with higher risks among patients havingcomplications of pseudoaneurysms, failure to successfully treat suchpseudoaneurysms, blood loss, transfusions, extensive operations tocorrect the problems and arterial infections (See Sprouse et al. Themanagement of peripheral vascular complications associated with the useof percutaneous suture-mediated closure devices. J Vasc Surg. 2001April; 33(4):688-693.). Moreover, some of these complications can bedeadly, particularly in patients with diabetes, obesity and previouslyimplanted devices (all conditions commonly found in patients in whomsuch closure devices are frequently used) (See Hollis and Retiring.Femoral endarteritis associated with percutaneous suture closure: newtechnology, challenging complications. J Vasc Surg. 2003 July;38(1):83-7.). Accordingly, there remains a great need to develop avascular closure system that avoids the problems associated with use ofknown vascular closure devices.

Another medical situation involving treatment of injured internal tissueis the repair of herniations. There are numerous types and locations ofhernia, and the surgical repair techniques vary widely dependingthereon. Both open and endoscopic procedures are currently in use, andmay involve the use of sutures alone or sutures in combination withvarious kinds of meshes or supports for the injured tissue. Majorcomplications for most hernia repair procedures include pain and therequirement to re-do the repair (See American College of Surgeons. Whenyou need an operation . . . About Hernia Repair, available at:http://www.facs.org/public info/operation/hernrep.pdf).

Similarly, there is also a need to improve the therapeutic options fortreatment of simple bleeding conditions such as epistaxis, whichrequires professional medical treatment in 1 of 7 people in theirlifetime (See Evans: Epistaxis, emedicine (2007) available atwww.emedicine.com/EMERG/topic806.htm). In fact, epistaxis is frequentlycited as the most common ENT emergency (See Hussain et al: Evaluation ofaetology and efficacy of management protocols of epistaxis. Ayub MedColl Abottabad, 2006 October-December; 18(4):63-6) The difficulty intreating these cases is evidenced by the fact that 1.6 out of every10,000 patients are hospitalized for epistaxis that is refractory tonormal treatment (See Viehweg et al: Epistaxis: diagnosis and treatment,J. Oral Maxillofac Surg 2006 March; 64(3):5 11-8). Current treatmentoptions include packing, chemical cauterization, electrocautery,surgical ligation and embolization (See: Ortiz & Bhattacharyya:Management pitfalls in the use of embolization for the treatment ofsevere epistaxis, Ear Nose Throat J. 2002 March; 82(3):178-83.)Frequently, multiple treatments with different technologies are requiredto effectively treat this often life-threatening condition (SeeSiniluoto et al: Embolization for the treatment of posterior epistaxis.An analysis of 31 cases. Arch Otolaryngol Head Neck Surg. August;119(8):837-41; Gifford & Orlandi: Epistaxis. Otoloaryngol Clin North Am.2008 June; 41(3):525-36, vii).

There are now in use a number of newer haemostatic agents that have beendeveloped to overcome the deficiencies of traditional gauze bandages.These haemostatic agents include the following:

-   -   Microporous polysaccharide particles (TraumaDEX®, Medafor Inc.,        Minneapolis, Minn.);    -   Zeolite (QuikClot®, Z-Medica Corp, Wallington, Conn.);    -   Acetylated poly-N-acetyl glucosamine (Rapid Deployment Hemostat™        (RDH), Marine Polymer Technologies, Danvers, Mass.);    -   Chitosan (HemCon® bandage, HemCon Medical Technologies inc.,        Portland Oreg.);    -   Liquid Fibrin Sealants (Tisseel V H, Baxter, Deerfield, Ill.)    -   Human fibrinogen and thrombin on equine collagen (TachoComb-S,        Hafslund Nycomed Pharma, Linz, Austria);    -   Microdispersed oxidized cellulose (m•doc™, Alltracel Group,        Dublin, Ireland);    -   Propyl gallate (Hemostatin™, Analytical Control Systems Inc.,        Fishers, Ind.);    -   Epsilon aminocaproic acid and thrombin (Hemarrest™ patch,        Clarion Pharmaceuticals, Inc);    -   Purified bovine corium collagen (Avitene® sheets (non-woven web        or Avitene Microfibrillar Collagen Hemostat (MCH), Davol, Inc.,        Cranston, R.I.);    -   Controlled oxidation of regenerated cellulose (Surgicel®,        Ethicon Inc., Somerville, N.J.);    -   Aluminum sulfate with an ethyl cellulose coating (Sorbastace        Microcaps, Hemostace, LLC, New Orleans, La.);    -   Microporous hydrogel-forming polyacrylamide (BioHemostat, Hemo        dyne, Inc., Richmond Va.); and    -   Recombinant activated factor VII (NovoSeven®, NovoNordisk Inc.,        Princeton, N.J.).        These agents have met with varying degrees of success when used        in animal models of traumatic injuries and/or in the field, and        with limited success in the sealing of therapeutic vascular        injuries.

Liquid fibrin sealants, such as Tisseel VII, have been used for years asan operating room adjunct for hemorrhage control. See J. L. Garza etal., J. Trauma 30:512-513 (1990); H. B. Kram et al, J. Trauma 30:97-101(1990); M. G. Ochsner et al, J. Trauma 30:884-887 (1990); T. L. Matthewet al., Ann Thorac. Surg. 50:40-44 (1990); H. Jakob et al., J. Vasc.Surg., 1:171-180 (1984). The first mention of tissue glue used forhemostasis dates back to 1909. See Current Trends in Surgical TissueAdhesives: Proceedings of the First International Symposium on SurgicalAdhesives, M. J. MacPhee et al., eds. (Lancaster, Pa.: TechnomicPublishing Co; 1995). Liquid fibrin sealants are typically composed offibrinogen and thrombin, but may also contain Factor XIII/XIIIa, eitheras a by-product of fibrinogen purification or as an added ingredient (incertain applications, it is therefore not necessary that FactorXIII/Factor XIIIa be present in the fibrin sealant because there issufficient Factor XIII/XIIIa, or other transaminase, endogenouslypresent to induce fibrin formation). As liquids, however, these fibrinsealants have not proved useful outside certain specific procedures.

Dry fibrinogen-thrombin dressings having a collagen support (e.g.TachoComb™, TachoComb™ H and TachoSil available from Hafslund NycomedPharma, Linz, Austria) are also available for operating room use in manyEuropean countries. See U. Schiele et al., Clin. Materials 9:169-177(1992). While these fibrinogen-thrombin dressings do not require thepre-mixing needed by liquid fibrin sealants, their utility for fieldapplications is limited by a requirement for storage at 4° C. and thenecessity for pre-wetting with saline solution prior to application tothe wound. These dressings are also not effective against high pressure,high volume bleeding. See Sondeen et al., J. Trauma 54:280-285 (2003).

A dry fibrinogen/thrombin dressing for treating wounded tissue is alsodisclosed in U.S. Pat. No. 6,762,336. This particular dressing iscomposed of a backing material and a plurality of layers, the outer twoof which contain fibrinogen (but no thrombin) while the inner layercontains thrombin and calcium chloride (but no fibrinogen). While thisdressing has shown great success in several animal models of hemorrhage,the bandage is fragile, inflexible, and has a tendency to break apartwhen handled. See McManus et al., Business Briefing: Emergency MedicalReview 2005, at 78.; Kheirabadi et al., J. Trauma 59:25-35 (2005). Inaddition, U.S. Pat. No. 6,762,336 teaches that this bandage shouldcontain 15 mg/cm² of fibrinogen to successfully pass a porcinearteriotomy test that is less robust than that disclosed in thisapplication (see Example XI). Moreover, although U.S. Pat. No. 6,762,336discloses that bandages comprising two layers of fibrinogen, each with aconcentration of 4 mg/cm² to 15 mg/cm² may provide effective control ofhemorrhage, it further teaches that “fibrinogen dose is related toquality. The higher dose is associated with more firm and tightlyadhered clots. While lower fibrinogen doses are effective for hemorrhagecontrol during the initial 60 minutes, longer term survival will likelydepend on clot quality.”

Other fibrinogen/thrombin-based dressings have also been proposed. Forexample, U.S. Pat. No. 4,683,142 discloses a resorptive sheet materialfor closing and healing wounds which consists of a glycoprotein matrix,such as collagen, containing coagulation proteins, such as fibrinogenand thrombin. U.S. Pat. No. 5,702,715 discloses a reinforced biologicalsealant composed of separate layers of fibrinogen and thrombin, at leastone of which also contains a reinforcement filler such as PEG, PVP, BSA,mannitol, FICOLL, dextran, myo-inositol or sodium chlorate. U.S. Pat.No. 6,056,970 discloses dressings composed of a bioabsorbable polymer,such as hyaluronic acid or carboxymethylcellulose, and a haemostaticcomposition composed of powdered thrombin and/or powdered fibrinogen.U.S. Pat. No. 7,189,410 discloses a bandage composed of a backingmaterial having thereon: (i) particles of fibrinogen; (ii) particles ofthrombin; and (iii) calcium chloride. U.S. Patent ApplicationPublication No. US 2006/0155234 A1 discloses a dressing composed of abacking material and a plurality of fibrinogen layers which havediscrete areas of thrombin between them. To date, none of thesedressings have been approved for use or are available commercially.

A number of different techniques, including the use of liquid fibrinsealant, have been proposed for sealing the punctures in blood vesselsmade to secure vascular access. For example, U.S. Pat. No. 7,357,794discloses devices, systems and methods for acute or chronic delivery ofsubstances or apparatus to extravascular treatment sites. U.S. Pat. No.7,335,220 discloses apparatus and methods for sealing a vascularpuncture using an expanding lyophylized hydrogel plug. U.S. Pat. No.7,300,663 discloses adhesion and sealing of tissue with compositionscontaining polyfunctional crosslinking agents and protein polymers. U.S.Pat. No. 7,399,483 discloses a carrier with solid fibrinogen and solidthrombin;. U.S. Pat. No. 7,335,220 discloses apparatus and methods forsealing vascular punctures. U.S. Pat. No. 7,115,588 discloses methodsfor treating a breach or puncture in a blood vessel. U.S. Pat. No.7,008,442 discloses vascular sealant delivery devices using liquidformulations. U.S. Pat. No. 6,890,342 discloses to methods and apparatusfor closing vascular puncture using a guidewire and/or other surgicalimplement extending from the wound on which a haemostatic material ismoved into contact with an area of the blood vessel surrounding thewound. U.S. Pat. No. 6,818,008 discloses percutaneous puncture sealingmethod using flowable sealants U.S. Pat. No. 6,699,262 discloses apercutaneous tissue track closure assembly and method using flowablematerials. U.S. Pat. No. 6,613,070 discloses sealing vascularpenetrations with haemostatic gels. U.S. Pat. No. 6,500,152 discloses adevice for introducing a two-component liquid fibrin adhesive into apuncture channel. U.S. Pat. No. 6,325,789 also discloses a device forsealing puncture wounds using liquid or paste fibrin sealant. U.S. Pat.No. 5,814,066 discloses methods of reducing femoral arterial bleedingusing percutaneous application of liquid fibrin sealant. U.S. Pat. No.5,725,551, U.S. Pat. No. 5,486,195 and U.S. Pat. No. 5,443,481 eachdisclose the use of two component liquid fibrin sealant for arteryclosure. U.S. Pat. No. 5,649,959 discloses an assembly for sealing apuncture in a vessel which maintains the fibrinogen and thrombinseparately. To date, however, all of these remain little-used intherapy, most likely due to the difficult and time consuming preparationrequirements for two-component liquid fibrin sealant compositions.

Similarly, two component liquid fibrin sealants have been used to attachsurgical meshes in the treatment of abdominal hernias. The surgicalresults have been excellent, typically as good or better than theefficacy of suture and staple fixation, with reduced complications andpost-operative pain. (See Schwab et al., Hernia. 2006 June; 10(3):272-7)

Liquid fibrin sealant has also be used to treat epistaxis, endoscopicsinus surgery and endonasal surgery ((See Vaiman et al. Fibrin gluetreatment for epistaxis. Rhinology. 2002 June; 40(2):99-91; Vaiman etal. Use of fibrin glue as a haemostatic in endoscopic sinus surgery. AnnOtol Rhinol Laryngol, 2005 March; 114(3): 237-41; Vaiman et al. Fibrinsealant: alternative to nasal packing in endonasal operations. Aprospective randomized study. Isr Med Assoc J. 2005 September;7(9):571-4.). All these reports indicate that liquid fibrin sealant maybe used with some success at controlling hemorrhage from variouslocations just inside the nose all the way into the sinuses. However,the time and efforts associated with preparing such sealants make themless than ideal for daily clinical use

Accordingly, there remains a need in the art for solid dressings thatcan be used to achieve hemostasis and sealing of internal woundedtissue, particularly highly vascularized tissue, and single bloodvessels. Additionally, treatment of tissues that have been divided (e.g.due to accident, pathology or surgical intervention) and requirere-approximation to promote healing would also benefit from a soliddressing capable of adequate tissue sealing.

The assessment of such dressings requires new techniques that go beyondthose previously disclosed for testing haemostatic dressings. Theability of dressings to seal an injured blood vessel has been determinedby an ex vivo porcine arteriotomy (EVPA) performance test, which wasfirst described in U.S. Pat. No. 6,762,336. The EVPA performance testevaluates the ability of a dressing to stop fluid flow through a hole ina porcine artery. While the procedure described in U.S. Pat. No.6,762,336 has been shown to be useful for evaluating haemostaticdressings, it failed to replicate faithfully the requirements forsuccess in vivo. More specifically, the procedure disclosed in U.S. Pat.No. 6,762,336 required testing at 37° C., whereas, in the real world,wounds are typically cooler than that. This decreased temperature cansignificantly reduce the rate of fibrin formation and its haemostaticefficacy in trauma victims. See, e.g., Acheson et al., J. Trauma59:865-874 (2005). The test in U.S. Pat. No. 6,762,336 also failed torequire a high degree of adherence of the dressing to the injuredtissue. A failure mode in which fibrin forms but the dressing fails toattach tightly to the tissue would, therefore, not be detected by thistest. Additionally, the pressure utilized in the procedure (200 mHg) maybe exceeded during therapy for some trauma patients. The overall resultof this is that numerous animal tests, typically involving small animals(such as rats and rabbits), must be conducted to accurately predictdressing performance in large animal, realistic trauma studies and inthe clinical environment.

In order to minimize the amount of time and the number of animal studiesrequired to develop dressings intended to treat accessible traumaticinjuries, an improved ex vivo testing procedure has been developed. Toaccomplish this, the basic conditions under which the dressing test wasconducted were changed, and the severity of the test parameters wasincreased to include testing at lower temperatures (i.e. 29-33° C. vs.37° C., representing the real physiologic challenge at realistic woundtemperatures (Acheson et al., J. Trauma 59:865-874 (2005)), higherpressures (i.e. 250 mmHg vs. 200 mmHg), a longer test period (3 minutesvs. 2 minutes) and larger sized arterial injuries (U.S. Pat. No.6,762,336 used an 18 gauge needle puncture, whereas the revisedprocedure used puncture holes ranging from 2.8 mm to 4 mm×6 mm). A newtest has also been developed to directly measure adherence of thedressing to the injured tissue. Both these tests showed greatly improvedstringency and are thus capable of surpassing the previous ex vivo testand replacing many in vivo tests for efficacy. These newer tests aredescribed in U.S. patent application Ser. No. 11/882,874, the disclosureof which is herein incorporated by reference in its entirety.

The newer tests described in U.S. patent application Ser. No. 11/882,874were designed to simulate trauma-derived, accessible wounds with highpressure and flow characteristics. Therefore, for the evaluation ofmethods and compositions for treating wounded internal tissue, it waspreferable to develop additional assays to more accurately simulate theperipheral vasculature and the effects of surrounding tissue.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide soliddressings that can treat wounded internal mammalian tissue. It isfurther an object of the present invention to provide a method oftreating wounded internal mammalian tissue, particularly human tissue.Other objects, features and advantages of the present invention will beset forth in the detailed description of preferred embodiments thatfollows, and will in part be apparent from that description and/or maybe learned by practice of the present invention. These objects andadvantages will be realized and attained by the compositions and methodsdescribed in this specification and particularly pointed out in theclaims that follow.

In accordance with these and other objects, a first embodiment of thepresent invention is directed to a method for treating wounded internaltissue in a mammal comprising applying to wounded internal tissue atleast one haemostatic material consisting essentially of a fibrinogencomponent and a fibrinogen activator for a time sufficient to reduce theflow of fluid from the wounded tissue, wherein the haemostatic materialis substantially homogeneous.

Another embodiment is directed to a method for treating wounded internaltissue in a mammal comprising applying to wounded internal tissue atleast one haemostatic material consisting essentially of a fibrinogencomponent and a fibrinogen activator for a time sufficient to reduce theflow of fluid from the wounded tissue, wherein the haemostatic materialis cast or formed from a single aqueous solution containing thefibrinogen component and the fibrinogen activator.

Another embodiment is directed to a method for treating wounded internaltissue in a mammal comprising applying to wounded internal tissue atleast one haemostatic material consisting essentially of a fibrinogencomponent and a fibrinogen activator for a time sufficient to reduce theflow of fluid from the wounded tissue, wherein the haemostatic materialis cast or formed as a single piece.

Still other embodiments of the invention are directed to the varioussolid and frozen haemostatic materials useful in the inventive methods.

It is to be understood that the foregoing general description and thefollowing detailed description of preferred embodiments are exemplaryand explanatory only and are intended to provide further explanation,but not limitation, of the invention as claimed herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram of the set-up for the ex vivo porcine carotidarteriotomoy assay described herein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsmentioned herein are incorporated by reference.

As used herein, use of a singular article such as “a,” “an,” and “the”is not intended to excluded pluralities of the article's object unlessthe context clearly and unambiguously dictates otherwise.

“Patient” as used herein refers to human or animal individuals in needof medical care and/or treatment.

“Wound” or “wounded tissue” as used herein refers to any damage to anyinternal tissue of a patient which results in the loss of blood from thecirculatory system and/or any other fluid from the patient's body. Thetissue may be any mammalian internal tissue, such as an organ or bloodvessel. A wound may be in a soft internal tissue, such as an organ, orin hard internal tissue, such as bone. The “damage” may have been causedby any agent or source, including traumatic injury, infection orsurgical intervention. Thus, the “damage” being treated according to themethods of the present invention may be the result of either an accidentor an intentional act.

“Resorbable material” as used herein refers to a substance that isbroken down spontaneously and/or by the mammalian body into componentswhich are consumed or eliminated in such a manner as not to interferesignificantly with wound healing and/or tissue regeneration, and withoutcausing any significant metabolic disturbance.

“Stability” as used herein refers to the retention of thosecharacteristics of a substance that determine activity and/or function.

“Suitable” as used herein is intended to mean that a substance (ormixture of substances) does not adversely affect the stability of thedressings or any component thereof.

“Binding agent” as used herein refers to a compound or mixture ofcompounds that improves the adherence and/or cohesion of the componentsof the haemostatic material of the dressings.

“Solubilizing agent” as used herein refers to a compound or mixture ofcompounds that improves the dissolution of a protein or proteins inaqueous solvent.

“Filler” as used herein refers to a compound or mixture of compoundsthat provide bulk and/or porosity to the haemostatic material.

“Release agent” as used herein refers to a compound or mixture ofcompounds that facilitates removal of a dressing from a manufacturingmold.

“Foaming agent” as used herein refers to a compound or mixture ofcompounds that produces gas when hydrated under suitable conditions.

“Solid” as used herein is intended to mean that a haemostatic materialor dressing will not substantially change in shape or form when placedon a rigid surface and then left to stand at room temperature for 24hours.

“Frozen” as used herein is intended to mean that a haemostatic materialor dressing will not substantially change in shape or form when placedon a rigid surface and then left to stand at 0° C. for 24 hours, butwill substantially change in shape or form when placed on a rigidsurface and then left at room temperature for 24 hours. Thus, in thecontext of the present invention, a “solid” dressing is not “frozen” anda “frozen” composition is not “solid”.

“Substantially homogeneous” as used herein is intended to mean that thehaemostatic material has a uniform composition throughout, within thetolerances described herein. Thus, a “substantially homogeneous”haemostatic material according to the present invention may be composedof a plurality of particles, provided that each of those particles hasthe same composition.

A first preferred embodiment of the present invention is directed to amethod for treating wounded internal tissue in a mammal comprisingapplying to wounded internal tissue at least one haemostatic materialconsisting essentially of a fibrinogen component and a fibrinogenactivator for a time sufficient to reduce the flow of fluid from thewounded tissue, wherein the haemostatic material is substantiallyhomogeneous.

Another embodiment is directed to a method for treating wounded internaltissue in a mammal comprising applying to wounded internal tissue atleast one haemostatic material consisting essentially of a fibrinogencomponent and a fibrinogen activator for a time sufficient to reduce theflow of fluid from the wounded tissue, wherein the haemostatic materialis cast or formed from a single aqueous solution containing thefibrinogen component and the fibrinogen activator.

Another embodiment is directed to a method for treating wounded internaltissue in a mammal comprising applying to wounded internal tissue atleast one haemostatic material consisting essentially of a fibrinogencomponent and a fibrinogen activator for a time sufficient to reduce theflow of fluid from the wounded tissue, wherein the haemostatic materialis cast or formed as a single piece.

Another preferred embodiment is directed to a frozen haemostaticmaterial for treating wounded internal tissue in a mammal consistingessentially of a fibrinogen component and a fibrinogen activator.

As used herein, “consisting essentially of” is intended to mean that thefibrinogen component and the fibrinogen activator are the only necessaryand essential ingredients of the haemostatic material when it is used asintended to treat wounded internal tissue. Accordingly, the haemostaticmaterial may contain other ingredients in addition to the fibrinogencomponent and the fibrinogen activator as desired for a particularapplication, but these other ingredients are not required for the soliddressing to function as intended under normal conditions, i.e. theseother ingredients are not necessary for the fibrinogen component andfibrinogen activator to react and form enough fibrin to reduce the flowof blood and/or fluid from normal wounded tissue when that dressing isapplied to that tissue under the intended conditions of use. If,however, the conditions of use in a particular situation are not normal,for example the patient is a hemophiliac suffering from Factor XIIIdeficiency, then the appropriate additional components, such as FactorXIII/XIIIa or some other transaminase, may be added to the haemostaticmaterial without deviating from the spirit of the present invention.

According to certain embodiments of the present invention, thehaemostatic material is formed or cast as a single piece. Once such isformed or cast, the haemostatic material may then be used as is or itmay be further processed, for example by grinding into a powder ofpre-determined particle size. Such particles may then be used as is ormay be combined with other substances for a particular application, e.g.such particles of haemostatic material may be mixed with a foaming agentor aerosol gas or may be combined with one or more binding agents andapplied to a support material.

The haemostatic materials of the present invention may be formed or castin any shape or form suitable for a given application. For example, thehaemostatic material may be formed or cast in the shape of a cone orcylinder or the like. Such a shape is particularly suitable for use inapplications where the damage to the tissue being treated is a hole tobe plugged or sealed, .e.g a vein which has been intentionally puncturedas part of a medical procedure, such as angioplasty. In suchapplications, the haemostatic material may alternatively be in the shapeof a disk, optionally with a hole for use in conjunction with a guidewire. Additionally, each of these forms can also be prepared bycombining particles of the inventive haemostatic materials with at leastone suitable binding agent in an appropriate mold.

The haemostatic material may also be formed or cast in the shape of aflat sheet. Such a form is particularly suitable for use in applicationswhere tissue needs to be sealed or approximated, for example inconnection with endoscopic surgery or, hernia repair. Alternatively, aflat sheet may be prepared by combining particles of the inventivehaemostatic materials with one or more suitable binding agents,optionally in a mold.

The haemostatic material may also optionally contain one or moresuitable fillers, such as sucrose, lactose, maltose, silk, fibrin,collagen, albumin (natural or recombinantly produced), polysorbate(Tween™), chitin, chitosan and its derivatives (e.g. NOCC-chitosan),alginic acid and salts thereof, cellulose and derivatives thereof,proteoglycans, hyaluron and its derivatives, such as hyaluronic acid,glycolic acid polymers, lactic acid polymers, glycolic acid/lactic acidco-polymers, and mixtures of two or more thereof.

The haemostatic material may also optionally contain one or moresuitable solubilizing agents, including detergents and tensides.Illustrative examples of suitable solubilizing agents include, but arenot limited to, the following: sucrose, dextrose, mannose, trehalose,mannitol, sorbitol, albumin, hyaluron and its derivatives, such ashyaluronic acid, sorbate, polysorbate (Tween™), sorbitan (SPAN™) andmixtures of two or more thereof.

The haemostatic material may also optionally contain one or moresuitable foaming agents, such as a mixture of a physiologicallyacceptable acid (e.g. citric acid or acetic acid) and a physiologicallysuitable base (e.g. sodium bicarbonate or calcium carbonate). Othersuitable foaming agents include, but are not limited to, dry particlescontaining pressurized gas, such as sugar particles containing carbondioxide (see, e.g., U.S. Pat. No. 3,012,893) or other physiologicallyacceptable gases (e.g. Nitrogen or Argon), and pharmacologicallyacceptable peroxides. Such a foaming agent may be introduced into theaqueous mixture of the fibrinogen component and the fibrinogenactivator, or may be introduced into an aqueous solution of thefibrinogen component and/or an aqueous solution of the fibrinogenactivator prior to mixing. Alternatively, the inventive haemostaticmaterials may be ground to particles of a predetermined size and thencombined with a suitable foaming agent.

The haemostatic material may also optionally contain a suitable sourceof calcium ions, such as calcium chloride, and/or a fibrin cross-linker,such as a transaminase (e.g. Factor XIII/XIIIa) or glutaraldehyde.

The haemostatic materials of the present invention are most preferablyprepared by mixing aqueous solutions of the fibrinogen component and thefibrinogen activator under conditions which minimize the activation ofthe fibrinogen component by the fibrinogen activator. This aqueousmixture of the fibrinogen component and the fibrinogen activator maythen be frozen until used to treat wounded tissue. Alternatively, themixture may then subjected to a process, such as lyophilization orfreeze-drying, to reduce the moisture content to a predeterminedeffective level, i.e. to a level where the dressing is solid andtherefore will not substantially change in shape or form upon standingat room temperature for 24 hours. Similar processes that achieve thesame result, such as drying, spray-drying, vacuum drying andvitrification, may also be employed, either alone or in combination.

As used herein, “moisture content” refers to levels determined byprocedures substantially similar to the FDA-approved, modified KarlFischer method (Centers for Biologics Evaluation and Research, FDA,Docket No. 89D-0140, 83-93; 1990 and references cited therein) or bynear infrared spectroscopy. Suitable moisture content(s) for aparticular inventive haemostatic material may be determined empiricallyby one skilled in the art depending upon the intended application(s)thereof.

For example, in certain embodiments of the present invention, highermoisture contents are associated with more flexible solid dressings.Thus, in solid dressings intended to be deformed in use, it may bepreferred for the haemostatic material to have a moisture content of atleast 6% and even more preferably in the range of 6% to 44%.

Similarly, in other embodiments of the present invention, lower moisturecontents are associated with more rigid solid dressings. Thus, in soliddressings intended to be used as formed or cast, it may be preferred forthe haemostatic material to have a moisture content of less than 6% andeven more preferably in the range of 1% to 6%.

Accordingly, illustrative examples of suitable moisture contents for theinventive haemostatic materials include, but are not limited to, thefollowing (each value being ±0.9%): less than 53%; less than 44%; lessthan 28%; less than 24%; less than 16%; less than 12%; less than 6%;less than 5%; less than 4%; less than 3%; less than 2.5%; less than 2%;less than 1.4%; between 0 and 12%, non-inclusive; between 0 and 6%;between 0 and 4%; between 0 and 3%; between 0 and 2%; between 0 and 1%;between 1 and 16%; between 1 and 11%; between 1 and 8%; between 1 and6%; between 1 and 4%; between 1 and 3%; between 1 and 2%; and between 2and 4%.

The fibrinogen component in the haemostatic material may be any suitablefibrinogen known and available to those skilled in the art. Thefibrinogen component may also be a functional derivative or metaboliteof a fibrinogen, such the fibrinogen α, β and/or γ chains, solublefibrin I or fibrin II, or a mixture of two or more thereof. A specificfibrinogen (or functional derivative or metabolite) for a particularapplication may be selected empirically by one skilled in the art. Asused herein, the term “fibrinogen” is intended to include mixtures offibrinogen and small mounts of Factor XIII/Factor XIIIa, or some othersuch transaminase. Such small amounts are generally recognized by thoseskilled in the art as usually being found in mammalian fibrinogen afterit has been purified according to the methods and techniques presentlyknown and available in the art, and typically range from 0.1 to 20Units/mL.

Preferably, the fibrinogen employed as the fibrinogen component is apurified fibrinogen suitable for introduction into a mammal. Typically,such fibrinogen is a part of a mixture of human plasma proteins whichinclude Factor XIII/XIIIa and have been purified to an appropriate leveland virally inactivated. A preferred aqueous solution of fibrinogen forpreparation of a solid dressing contains around 37.5 mg/mL fibrinogen ata pH of around 7.4±0.1. Suitable fibrinogen for use as the fibrinogencomponent has been described in the art, e.g. U.S. Pat. No. 5,716,645,and similar materials are commercially available, e.g. from sources suchas Sigma-Aldrich, Enzyme Research Laboratories, HaematologicTechnologies and Aniara.

The fibrinogen component should be present in the inventive haemostaticmaterials in an amount effective to react with the fibrinogen activatorand form sufficient fibrin to reduce the flow of fluid from woundedinternal tissue. According to certain preferred embodiments of thepresent invention, when the haemostatic material is frozen, thefibrinogen component is present in an amount of from 4.70 mg to 18.75 mg(±0.009 mg) per square centimeter of the surface(s) of the haemostaticmaterial intended to contact the wounded internal tissue. *******

According to other preferred embodiments, when the haemostatic materialis a solid, regardless of form, the fibrinogen component is present inan amount of from 5.00 mg to 450.00 mg (±0.009 mg) per square centimeterof the surface(s) intended to contact the wounded internal tissue beingtreated. Greater or lesser amounts, however, may be employed dependingupon the particular application intended for the solid dressing.

For example, when the haemostatic material is in the shape of a rod orcylinder, the fibrinogen component is more preferably present in anamount of from 25.00 mg to 75.00 mg (±0.009 mg) per square centimeter ofthe surface(s) intended to contact the wounded internal tissue beingtreated. Alternatively, when the haemostatic material is in the shape ofa flat sheet or disk, the fibrinogen component is more preferablypresent in an amount of from 5.00 to 56.00 mg (±0.009 mg) per squarecentimeter of the surface(s) intended to contact the wounded internaltissue being treated. Still alternatively, when the haemostatic materialis powdered, either loose or compressed, the fibrinogen component ismore preferably present in an amount from 26.00 mg to 450.00 mg (±0.09mg) per square centimeter of the surface(s) intended to contact thewounded internal tissue being treated.

The fibrinogen activator employed in the haemostatic materials of thepresent invention may be any of the substances or mixtures of substancesknown by those skilled in the art to convert fibrinogen (or a fibrinogenequivalent) into fibrin. Illustrative examples of suitable fibrinogenactivators include, but are not limited to, the following: thrombins,such as human thrombin or bovine thrombin, and prothrombins, such ashuman prothrombin or prothrombin complex concentrate (a mixture ofFactors II, VII, IX and X); snake venoms, such as batroxobin, reptilase(a mixture of batroxobin and Factor XIIIa), bothrombin, calobin,fibrozyme, and enzymes isolated from the venom of Bothrops jararacussu;and mixtures of any two or more of these. See, e.g., Dascombe et al.,Thromb. Haemost. 78:947-51 (1997); Hahn et al., J. Biochem, (Tokyo)119:835-43 (1996); Fortova et al., J. Chromatogr. S. Biomed. Appl.694:49-53 (1997); and Andriao-Escarso et al., Toxicon. 35: 1043-52(1997).

Preferably, the fibrinogen activator is a thrombin. More preferably, thefibrinogen activator is a mammalian thrombin, although bird and/or fishthrombin may also be employed in appropriate circumstances. While anysuitable mammalian thrombin may be used, the thrombin employed ispreferably a lyophilized mixture of human plasma proteins which has beensufficiently purified and virally inactivated for the intended use ofthe solid dressing. Suitable thrombin is available commercially fromsources such as Sigma-Aldrich, Enzyme Research Laboratories,Haematologic Technologies and Biomol International. A particularlypreferred aqueous solution of thrombin for preparing the inventivehaemostatic materials contains thrombin at a potency of between 10 and2000±50 International Units/mL, and more preferred at a potency of25±2.5 International Units/mL. Other constituents may include albumin(generally about 0.1 mg/mL) and glycine (generally about 100 mM±0.1 mM).The pH of this particularly preferred aqueous solution of thrombin isgenerally in the range of 6.5-7.8, and preferably 7.4±0.1, although a pHin the range of 5.5-8.5 may be acceptable.

In addition to the inventive haemostatic material(s), the solid andfrozen dressings of the present invention may optionally furthercomprise one or more support materials. As used herein, a “supportmaterial” refers to a material that sustains or improves the structuralintegrity of the solid or frozen dressing and/or the fibrin clot formedwhen such a dressing is applied to wounded tissue. The support materialmay be an internal support material or a surface support material.Moreover, in the case of the latter, if the dressing is in a form thathas a wound facing side, the support material may be on the wound facingside or it may be on the non-wound facing side or both.

Any suitable resorbable material known and available to those skilled inthe art may be employed in the present invention. For example, theresorbable material may be a proteinaceous substance, such as silk,fibrin, keratin, collagen and/or gelatin. Alternatively, the resorbablematerial may be a carbohydrate substance, such as alginates, chitin,cellulose, proteoglycans (e.g. poly-N-acetyl glucosamine), glycolic acidpolymers, lactic acid polymers, or glycolic acid/lactic acidco-polymers. The resorbable material may also comprise a mixture ofproteinaceous substances or a mixture of carbohydrate substances or amixture of both proteinaceous substances and carbohydrate substances.Specific resorbable material(s) may be selected empirically by thoseskilled in the art depending upon the intended use of the soliddressing.

According to certain preferred embodiments of the present invention, theresorbable material is a carbohydrate substance. Illustrative examplesof particularly preferred resorbable materials include, but are notlimited to, the materials sold under the trade names Vicryl™ (a glycolicacid/lactic acid copolymer) and Dexon™ (a glycolic acid polymer).

Any suitable non-resorbable material known and available to thoseskilled in the art may be employed as the support material. Illustrativeexamples of suitable non-resorbable materials include, but are notlimited to, plastics, silicone polymers, paper and paper products,latex, gauze plastics, non-resorbable suture materials, latexes andsuitable derivatives thereof.

According to other preferred embodiments, the support material comprisesan internal support material. Such an internal support material ispreferably fully contained within the haemostatic material(s) of a solidor frozen dressing. The internal support material may take any formsuitable for the intended application of the haemostatic material. Forexample, according to certain embodiments, the internal support materialmay be particles of a predetermined suitable size which are dispersedthroughout the haemostatic material. Alternatively, a sheet or film orinternal support material may be included in the solid or frozenhaemostatic material.

According to still other preferred embodiments, the support material maycomprise a backing material on the surface(s) of the dressing oppositethe wound-facing surface. As with the internal support material, thebacking material may be a resorbable material or a non-resorbablematerial, or a mixture thereof, such as a mixture of two or moreresorbable materials or a mixture of two or more non-resorbablematerials or a mixture of resorbable material(s) and non-resorbablematerial(s).

According to still other preferred embodiments, the dressing comprisesboth a backing material and an internal support material in addition tothe haemostatic material(s). According to still other preferredembodiments, the dressing comprises both a front support material and aninternal support material in addition to the haemostatic layer(s).According to still other preferred embodiments, the dressing comprises abacking material, a front support material and an internal supportmaterial in addition to the haemostatic layer(s).

According to certain preferred embodiments, the haemostatic material(s)may also contain a binding agent to maintain the physical integrity ofthe haemostatic material(s). Illustrative examples of suitable bindingagents include, but are not limited to, sucrose, mannitol, sorbitol,gelatin, hyaluron and its derivatives, such as hyaluronic acid, maltose,povidone, starch, chitosan and its derivatives, and cellulosederivatives, such as carboxymethylcellulose, as well as mixtures of twoor more thereof.

According to certain embodiments of the present invention, particularlywhere the solid or frozen dressing is manufactured using a mold, thedressings may also optionally further comprise a release layer inaddition to the haemostatic material(s) and support layer(s). As usedherein, a “release layer” refers to a layer containing one or moreagents (“release agents”) which promote or facilitate removal of thesolid or frozen dressing from a mold in which it has been manufactured.A preferred such agent is sucrose, but other suitable release agentsinclude gelatin, hyaluron and its derivatives, including hyaluronicacid, mannitol, sorbitol and glucose. Alternatively, such one or morerelease agents may be contained in the haemostatic material.

The haemostatic material and any layer(s) may be affixed to one anotherby any suitable means known and available to those skilled in the art.For example, a physiologically-acceptable adhesive may be applied to abacking material (when present), and the haemostatic materialsubsequently affixed thereto.

In certain embodiments of the present invention, thephysiologically-acceptable adhesive has a shear strength and/orstructure such that the backing material can be separated from thefibrin clot formed by the haemostatic layer after application of thedressing to wounded tissue. In other embodiments, thephysiologically-acceptable adhesive has a shear strength and/orstructure such that the backing material cannot be separated from thefibrin clot after application of the bandage to wounded tissue.

Suitable fibrinogen components and suitable fibrinogen activators forthe haemostatic materials may be obtained from any appropriate sourceknown and available to those skilled in the art, including, but notlimited to, the following: from commercial vendors, such asSigma-Aldrich and Enzyme Research Laboratories; by extraction andpurification from human or mammalian plasma by any of the methods knownand available to those skilled in the art; from supernatants or pastesderived from plasma or recombinant tissue culture, viruses, yeast,bacteria, or the like that contain a gene that expresses a human ormammalian plasma protein which has been introduced according to standardrecombinant DNA techniques; and/or from the fluids (e.g. blood, milk,lymph, urine or the like) of transgenic mammals (e.g. goats, sheep,cows) that contain a gene which has been introduced according tostandard transgenic techniques and that expresses the desired fibrinogenand/or desired fibrinogen activator.

According to certain preferred embodiments of the present invention, thefibrinogen component is a mammalian fibrinogen such as bovinefibrinogen, porcine fibrinogen, ovine fibrinogen, equine fibrinogen,caprine fibrinogen, feline fibrinogen, canine fibrinogen, murinefibrinogen or human fibrinogen. According to other embodiments, thefibrinogen component is bird fibrinogen or fish fibrinogen. According toany of these embodiments, the fibrinogen component may be recombinantlyproduced fibrinogen or transgenic fibrinogen.

According to certain preferred embodiments of the present invention, thefibrinogen activator is a mammalian thrombin, such as bovine thrombin,porcine thrombin, ovine thrombin, equine thrombin, caprine thrombin,feline thrombin, canine thrombin, murine thrombin and human thrombin.According to other embodiments, the thrombin is bird thrombin or fishthrombin. According to any of these embodiments, the thrombin may berecombinantly produced thrombin or transgenic thrombin.

As a general proposition, the purity of the fibrinogen component and/orthe fibrinogen activator for use in the solid dressing will be a purityknown to one of ordinary skill in the relevant art to lead to theoptimal efficacy and stability of the protein(s). Preferably, thefibrinogen component and/or the fibrinogen activator has been subjectedto multiple purification steps, such as precipitation, concentration,diafiltration and affinity chromatography (preferably immunoaffinitychromatography), to remove substances which cause fragmentation,activation and/or degradation of the fibrinogen component and/or thefibrinogen activator during manufacture, storage and/or use of the soliddressing. Illustrative examples of such substances that are preferablyremoved by purification include: protein contaminants, such asinter-alpha trypsin inhibitor and pre-alpha trypsin inhibitor;non-protein contaminants, such as lipids; and mixtures of protein andnon-protein contaminants, such as lipoproteins. The fibrinogen componentand/or fibrinogen activator and/or the inventive haemostatic materialsmay also be subjected to suitable sterilization treatments, including,but not limited to, treatment with one or more of the following: heat,gamma radiation, e-beam radiation, plasma radiation and ethylene oxide.

The amount of the fibrinogen activator employed in the solid dressing ispreferably selected to optimize both the efficacy and stability thereof.As such, a suitable concentration for a particular application of thesolid dressing may be determined empirically by one skilled in therelevant art.

According to certain preferred embodiments of the present invention,when the fibrinogen activator is human thrombin, the amount of humanthrombin employed is between 0.03 and 16.10 Units (all values being±0.009) per square centimeter of the surface(s) of the haemostaticmaterial intended to contact the wounded internal tissue. Greater orlesser amounts, however, may be employed depending upon the particularapplication intended for the solid dressing.

For example, when the haemostatic material is a solid in the shape of arod or cylinder, the fibrinogen activator is more preferably present inan amount of from 2.50 Units to 7.50 Units (±0.009 Units) per squarecentimeter of the surface(s) intended to contact the wounded internaltissue being treated. Alternatively, when the haemostatic material is asolid in the shape of a flat sheet or disk, the fibrinogen activator ismore preferably present in an amount of from 0.03 Units to 16.10 Units(±0.009 Units) per square centimeter of the surface(s) intended tocontact the wounded internal tissue being treated. Still alternatively,when the haemostatic material is a powdered solid, either loose orcompressed, the fibrinogen activator is more preferably present in anamount of about 1.3 Units (±0.09 mg) per square centimeter of thesurface(s) intended to contact the wounded internal tissue beingtreated. Still alternatively, when the haemostatic material is frozen,the fibrinogen activator is more preferably present in an amount ofabout 1.3 Units (±0.09 mg) pet square centimeter of the surface(s)intended to contact the wounded internal tissue being treated.

According to still other preferred embodiments of the present invention,when the fibrinogen activator is human thrombin, the amount of humanthrombin employed is between 0.0087 and 1.0000 Units (all values being±0.00009) per milligram of the fibrinogen component. Greater or lesseramounts, however, may be employed depending upon the particularapplication intended for the solid dressing.

For example, when the haemostatic material is a solid in the shape of arod or cylinder, the fibrinogen activator is more preferably present inan amount of about 0.1 Units (±0.09 Units) per milligram of thefibrinogen component. Alternatively, when the haemostatic material is asolid in the shape of a flat sheet or disk, the fibrinogen activator ismore preferably present in an amount of from 0.1 Units to 1.00 Units(±0.009 Units) per milligram of the fibrinogen component. Stillalternatively, when the haemostatic material is a powdered solid, eitherloose or compressed, the fibrinogen activator is more preferably presentin an amount of about 0.0087 Units to 0.0500 Units (±0.00009 Units) permilligram of the fibrinogen component. Still alternatively, when thehaemostatic material is frozen, the fibrinogen activator is morepreferably present in an amount of about 0.07 Units to 0.10 Units(±0.009 Units) per milligram of the fibrinogen component.

During use of the inventive haemostatic materials, the fibrinogencomponent and the fibrinogen activator are preferably activated at thetime the dressing is applied to the wounded tissue by the endogenousfluids of the patient escaping from the hemorrhaging wound.Alternatively, in situations where fluid loss from the wounded tissue isinsufficient to provide adequate hydration of the protein layers, thefibrinogen component and/or the fibrinogen activator may be activated bya suitable, physiologically-acceptable liquid, optionally containing anynecessary co-factors and/or enzymes, prior to or during application ofthe dressing to the wounded tissue.

In some embodiments of the present invention, the inventive haemostaticmaterials may also contain one or more supplements, such as growthfactors, drugs, polyclonal and monoclonal antibodies and othercompounds. Illustrative examples of such supplements include, but arenot limited to, the following: fibrinolysis inhibitors, such asaprotonin, tranexamic acid and epsilon-amino-caproic acid; antibiotics,such as tetracycline and ciprofloxacin, amoxicillin, and metronidazole;anticoagulants, such as activated protein C, heparin, prostacyclins,prostaglandins (particularly (PGI₂), leukotrienes, antithrombin III,ADPase, and plasminogen activator; steroids, such as dexamethasone,inhibitors of prostacyclin, prostaglandins, leukotrienes and/or kininsto inhibit inflammation; cardiovascular drugs, such as calcium channelblockers, vasodilators and vasoconstrictors, such as epinephrine;chemoattractants; local anesthetics such as bupivacaine; andantiproliferative/antitumor drugs such as 5-fluorouracil (5-FU), taxoland/or taxotere; antivirals, such as gangcyclovir, zidovudine,amantidine, vidarabine, ribaravin, trifluridine, acyclovir,dideoxyuridine and antibodies to viral components or gene products;cytokines, such as alpha- or beta- or gamma-Interferon, alpha- orbeta-tumor necrosis factor, and interleukins; colony stimulatingfactors; erythropoietin; antifungals, such as diflucan, ketaconizole andnystatin; antiparasitic gents, such as pentamidine; anti-inflammatoryagents, such as alpha-1-anti-trypsin and alpha-1-antichymotrypsin;anesthetics, such as bupivacaine; analgesics; antiseptics; hormones;vitamins and other nutritional supplements; glycoproteins; fibronectin;peptides and proteins; carbohydrates (both simple and/or complex);proteoglycans; antiangiogenins; antigens; lipids or liposomes;oligonucleotides (sense and/or antisense DNA and/or RNA); and genetherapy reagents. In other embodiments of the present invention, thebacking layer and/or the internal support layer, if present, may containone or more supplements. According to certain preferred embodiments ofthe present invention, the therapeutic supplement is present in anamount greater than its solubility limit in fibrin.

The inventive haemostatic materials, and the solid and frozen dressingscontaining them, may be applied to any internal wounded tissue in amammal using any of the suitable techniques and/or devices known andavailable to one skilled in the medical arts. For example, when used totreat vascular punctures, the haemostatic material(s) may be applied viaa catheter, either with or without a guide wire. The inventive materialsand dressings may also be applied in conjunction with endoscopictechniques, including endoscopic surgery, laparoscopic surgery andtele-robotic/tele-prescesne surgery. According to such embodiments, itis preferable to use a “plunger” or “tamper” to facilitate passage ofthe inventive materials through surrounding tissue to the woundedinternal tissue being treated. The inventive materials and dressings mayalso be applied manually.

The following examples are illustrative only and are not intended tolimit the scope of the invention as defined by the appended claims. Itwill be apparent to those skilled in the art that various modificationsand variations can be made in the methods of the present inventionwithout departing from the spirit and scope of the invention. Thus, itis intended that the present invention cover the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

EXAMPLES

The following is a list of acronyms used in the Examples below:

-   CFB: Complete Fibrinogen Buffer (100 mM Sodium Chloride, 1.1 mM    Calcium Chloride, 10 mM Tris, 10 mM Sodium Citrate, 1.5% Sucrose,    Human Serum Albumin (80 mg/g of total protein) and Tween™ 80    (non-animal source) 15 mg/g total protein)-   CTB: Complete Thrombin Buffer (150 mM Sodium Chloride, 40 mM Calcium    Chloride, 10 mM Tris and 100 mM L-Lysine with the addition of HSA at    100 ug/ml)-   ERL: Enzyme Research Laboratories-   EVPA: Ex Vivo Porcine Arteriotomy-   EVPCA: Ex Vivo Porcine Carotid Arteriotomy-   FD: Inventive haemostatic dressing-   HSA: Human Serum Albumin-   HD: A “sandwich” fibrin sealant haemostatic dressing as disclosed in    U.S. Pat. No. 6,762,336-   IFB: Incomplete Fibrinogen Buffer.; CFB without HSA and Tween-   Fibrinogen Dose In a solid mass, the amount of fibrinogen within the    mass divided by the surface area to be treated. Usually expressed in    mg of Fibrinogen per cm², where the mass of fibrinogen is determined    via a clottable protein assay-   PETG: Glycol-modified Polyethlylenetetrapthalate-   PPG: Polypropylene-   PVC: Poly vinyl chloride-   T:F Thrombin to Fibrinogen ratio. In a test article, the amount of    thrombin activity per unit of fibrinogen. Usually expressed in    thrombin NIH Units per mg of fibrinogen (measured via a clottable    protein assay)-   Thrombin Dose In a solid mass, the amount of thrombin within the    mass divided by the surface area to be treated. Usually expressed in    NIH Units of thrombin per cm²-   TRIS: trishydroxymethylaminomethane    (2-amino-2-hydroxymethyl-1,3-propanediol)

The ability of the dressings to seal an injured blood vessel wasdetermined by modifications of an ex vivo porcine arteriotomy (EVPA)performance test, which was first described in U.S. Pat. No. 6,762,336.The EVPA performance test evaluates the ability of a dressing to stopfluid flow through a hole in a porcine artery. While the proceduredescribed in U.S. Pat. No. 6,762,336 has been shown to be useful forevaluating haemostatic dressings, it failed to replicate faithfully therequirements for success in vivo. More specifically, the proceduredisclosed in U.S. Pat. No. 6,762,336 required testing at 37° C.,whereas, in the real world, wounds are typically cooler than that. Thisdecreased temperature can significantly reduce the rate of fibrinformation and its haemostatic efficacy in trauma victims. See, e.g.,Acheson et al., J. Trauma 59:865-874 (2005). The test in U.S. Pat. No.6,762,336 also failed to require a high degree of adherence of thedressing to the injured tissue. A failure mode in which fibrin forms butthe dressing fails to attach tightly to the tissue would, therefore, notbe detected by this test. Additionally, the pressure utilized in theprocedure (200 mHg) may be exceeded during therapy for some traumapatients. The overall result of this is that numerous animal tests,typically involving small animals (such as rats and rabbits), must beconducted to accurately predict dressing performance in large animal,realistic trauma studies and in the clinical environment.

In order to minimize the amount of time and the number of animal studiesrequired to develop the present invention, an improved ex vivo testingprocedure was developed. To accomplish this, the basic conditions underwhich the dressing test was conducted were changed, and the severity ofthe test parameters was increased to include testing at lowertemperatures (i.e. 29-33° C. vs. 37° C., representing the realphysiologic challenge at realistic wound temperatures (Acheson et al.,J. Trauma 59:865-874 (2005)), higher pressures (i.e. 250 mmHg vs. 200mmHg), a longer test period (3 minutes vs. 2 minutes) and larger sizedarterial injuries (U.S. Pat. No. 6,762,336 used an 18 gauge needlepuncture, whereas the revised procedure used puncture holes ranging from2.8 mm to 4 mm×6 mm).

In addition, a new test was derived to directly measure adherence of thedressing to the injured tissue.

Example 1

In order to apply the haemostatic test articles to the surface of aninjured artery surrounded by a tissue stimulant, the test articles werehoused in cylindrical molds made of 10 or 3 mL polypropylene syringes(Becton Dickinson) with the luer-lock end removed. The plungers werewithdrawn to the 6 mL and 2 mL mark respectively. For dressingsutilizing a backing, the support material was cut and placed into eachmold and pushed down until it was adjacent to the plunger. Once preparedthe molds were placed upright and surrounded by dry ice, leaving theopening exposed at the top. 1 ml of fibrinogen and 0.15 mL of thrombin(with or without backing material dispersed within) were dispensed intothe 10 mL molds and 1 ml of fibrinogen and 0.15 mL of thrombin (with orwithout support material dispersed within) were dispensed into the 3 mLmolds, which were allowed to freeze for 5 minutes. The molds were thenplaced into the −80° C. freezer for at least two hours before beingplaced into a pre-cooled Genesis™ lyophylizer (Virtis, Gardiner, N.Y.).The chamber was sealed and the temperature equilibrated. The chamber wasthen evacuated and the dressings lyophilized via a primary and secondarydrying cycle.

They were subsequently performance tested in a modified EVPA assay (DeepTissue EVPA). Briefly, in one version, a plastic foam form was slippedover the artery. This covering had a hole in it that corresponded to thehole in the artery and the surrounding tissue (FIG. 1). In anothervariant, the foam was replaced with a piece of tissue, specifically,bovine muscle, in which a hole had been prepared as with the foam. Thefoam was maintained at 37° C. by placement in a 37° C. water bath, whilethe muscle tissue was maintained at 37° C. by placement on a 37° C.block heater. Warm saline was added to the surface of the dressing andthe mold was immediately passed down thru the hole in the foam to theartery surface. The plunger was then depressed and held by hand for 3minutes, after which the mold was withdrawn as the plunger was depressedfurther. At this point the artery was pressurized and the assaycontinued as described hereafter.

Deep Tissue EVPA Testing

Equipment and Supplies:

-   -   In-line high pressure transducer (Ashcroft Duralife™ or        equivalent)    -   Peristaltic pump (Pharmacia Biotech™, Model P-1 or equivalent)    -   Voltmeter (Craftsman™ Professional Model 82324 or equivalent)    -   Computer equipped with software for recording pressure or        voltage information    -   Tygon™ tubing (assorted sizes) with attachments    -   Water bath (Baxter Durabath™ or equivalent), preset to 37° C.    -   Incubation chamber (VWR™, Model 1400G or equivalent), preset to        37° C.    -   Thermometer to monitor temperatures of both water bath and oven    -   Assorted forceps, hemostats, and scissors    -   10 cc. and 20 cc. syringes with an approximately 0.6 cm hole        drilled in center and smaller hole drilled through both syringe        and plunger. This hole, drilled into the end of the syringe,        will be used to keep the plunger drawn back and stationary.    -   O-rings (size 10 and 13)    -   Plastic Shields to fit the 10 cc and 20 cc syringes        (approximately 3.5 cm in length)    -   P-1000 Pipetman™ with tips    -   Programmable Logic Controller (PLC) to control the pumps to        maintain the desired pressure profile (Optional. Manual control        may be used if desired.)

1. Materials and Chemicals

-   -   Porcine descending aortas (Pel-Freez Biologicals™, Catalog        #59402-2 or equivalent)    -   Cyanoacrylate glue (Vetbond™, 3M or equivalent)    -   18-gauge needle(s)    -   0.9% Saline, maintained at 37° C.    -   Red food coloring    -   Vascular Punch(es), 2.8 mm or other    -   Plastic Wrap

2. Artery Cleaning and Storage

-   -   1. Store arteries at −20° C. until used.    -   2. Thaw arteries at 37° C. in H₂O bath.    -   3. Clean fat and connective tissue from exterior surface of        artery.    -   4. Cut the arteries into ˜5 cm segments.    -   5. The arteries may be refrozen to −20° C. and stored until use.

3. Artery Preparation for Assay

-   -   1. Turn the artery inside-out so that the smooth, interior wall        is facing outwards.    -   2. Stretch a size 13 O-ring over a 20 cc syringe or a size 10        O-ring over a 10 cc syringe with an approximately 0.6 cm (0.25        in) hole drilled into one side.    -   3. Pull the artery onto the syringe, taking care not to tear the        artery or have a too loose fit. The artery should fit snugly to        the syringe. Slide another O-ring of the same size onto the        bottom of the syringe    -   4. Carefully pull both O-rings over the ends of the artery. The        distance between the O-rings should be at least 3.5 cm    -   5. Using the blade of some surgical scissors, gently scrape the        surface of the artery in order to roughen the surface of the        artery.    -   6. Use a 18-gauge needle to poke a hole through the artery over        the site of the hole in the syringe barrel (see note above)    -   7. The tip of the biopsy punch is inserted through the hole in        the artery. Depress the punch's plunger to make an open hole in        the artery. Repeat a couple of times to ensure that the hole is        open and free of connective tissue.    -   8. Patch holes left by collateral arteries. Generally this is        done by cutting a patch from a latex glove and gluing it over        the hole with cyanoacrylate glue. Allow the glue to cure for at        least 10 minutes.        -   Place the artery in the warmed, moistened container and            place in the incubation chamber. Allow the arteries to warm            for at least 30 minutes.

4. Solution and Equipment Preparation

-   -   1. Check to see that the water bath, block heater and incubation        chamber are maintained at 37° C.    -   2. Make sure that there is sufficient 0.9% saline in the pump's        reservoir for completion of the day's assays. Add more if        needed.    -   3. Place 0.9% saline and 0.9% saline with a few drops of red        food coloring added into containers in a water bath so that the        solutions will be warmed prior to performing the assay.    -   4. Prepare the container for warming the arteries in the        incubation chamber by lining with KimWipes™ and adding a small        amount of water to keep the arteries moist.    -   5. Check the tubing for air bubbles. If bubbles exist, turn on        the pump and allow the 0.9% saline to flow until all bubbles are        removed.

5. Application of the Dressing

-   -   1. Slip either the warmed (at 37° C.) plastic foam form or the        warmed tissue over the artery. Align the hole in it to        correspond to the hole in the artery and the surrounding tissue        (FIG. 1).    -   2. Open the haemostatic dressing (Test Article) pouch and remove        haemostatic dressing & Applicator.    -   3. Slowly wet the haemostatic dressing drop wise with 0.9%        saline warmed to 29-33° C. or other blood substitute, taking        care to keep the saline from running off the edges. Any obvious        differences in wetting characteristics from the positive control        should be noted on the data collection forms.        -   NOTE: By way of example, a representative (13-15 mg/cm² of            fibrinogen) 2.4×2.4 cm haemostatic dressing should generally            be wet with 800 μl of saline or other blood substitute. The            amount of saline used can be adjusted depending on the            requirements of the particular experiment being performed;            however, any changes should be noted on the data collection            forms.    -   4. Immediately pass the dressing in the applicator down thru the        hole in the foam to the artery surface. Depress the plunger by        hand and hold by hand for 3 minutes, after which the applicator        is withdrawn as the plunger was depressed further.    -   5. After polymerization, note the condition of the haemostatic        dressing. Any variation from the positive control should be        noted on the data collection form.

EXCLUSION CRITERION: The mesh support material must remain over the holein the artery. If it has shifted during the polymerization and does notcompletely cover the hole the haemostatic dressing must be excluded.

Testing Procedure

1. Diagram of Testing Equipment Set-Up

The set-up of the testing equipment is shown in FIG. 1. Some additional,unshown components may be utilized to read out (pressure gauge) orcontrol the pressure within the system

2. Equipment and Artery Assembly

Fill the artery and syringe with red 0.9% saline warmed to 37° C.,taking care to minimize the amount of air bubbles within the syringe andartery. Filling the artery with the opening uppermost can assist withthis. Attach the artery and syringe to the testing apparatus, makingsure that there are as few air bubbles in the tubing as possible. Theperistaltic pump should be calibrated so that it delivers approximately3 ml/min. If available, the PLC should be operated according to apre-determined range of pressures and hold times as appropriate for thearticle being tested. If under manual control, the pressure/time profileto be followed is attained by manually turning the pump on and off whilereferencing the system pressure as read out by one or morepressure-reading components of the system. Following the conclusion oftesting, the haemostatic dressing is subjectively assessed with regardto adhesion to the artery and formation of a plug in the artery hole.Any variations from the positive control should be noted on the datacollection form.

Success Criteria

Haemostatic dressings that are able to withstand pressures for 3 minutesare considered to have passed the assay. When a haemostatic dressing hassuccessfully passed the assay the data collection should be stoppedimmediately so that the natural decrease in pressure that occurs in theartery once the test is ended isn't included on the graphs. Should theoperator fail to stop data collection, these points can be deleted fromthe data file to avoid confusing the natural pressure decay that occurspost-test with an actual dressing failure. The entire testing periodfrom application of the haemostatic dressing to completion must fallwithin pre-established criteria. The maximum pressure reached should berecorded on the data collection form.

Failure Criteria

Haemostatic dressings that start leaking saline at any point duringtesting are considered to have reached the end of the assay.

-   -   NOTE: Build failures that are caused by artery swelling can be        ignored and the test continued or re-started (as long as the        total testing time doesn't fall beyond the established limit).

When leakage does occur, the pressure should be allowed to fall ˜20 mmHgbefore data collection is stopped so that the failure is easily observedon the graphs. The pressures at which leakage occurred should berecorded on the data collection form. Should the data collection stop inthe middle of the experiment due to equipment failure the data can becollected by hand at 5 second intervals until the end of the test orhaemostatic dressing failure, whichever happens first. The data pointsshould be recorded on the back of the data collection form, clearlylabeled, and entered by hand into the data tables.

Exclusion Criteria

If the total testing period exceeds the maximum allowed for thatprocedure, regardless of cause, results must be excluded. If there areleaks from collaterals that can't be fixed either by patching or fingerpressure the results must be excluded. If the test fails because ofleaks at the O-rings, the results must be excluded. If the mesh supportmaterial does not completely cover the hole in the artery, the resultsmust be excluded.

Adherence Performance Testing

Equipment and Supplies

Hemostat(s), Porcine artery and haemostatic dressing, optionally afterperformance of EVPA assay.

Preparation of the Artery+Dressing

After application of the dressing without completion of the EVPA Assay,the dressing is ready for the Adherence Assay and Weight Limit Test (ifapplicable). After application of the dressing and subsequent EVPAAnalysis, the artery and syringe system is then disconnected slowly fromthe pump so that solution does not spray everywhere. The warmed, redsaline solution from the EVPA Assay remains in the syringe until theAdherence Assay and Weight Limit Test (if applicable) is completed.

Performance of the Adherence Assay

1. After preparation of the artery and dressing (with or without EVPAanalysis), gently lift the corner of the mesh and attach a hemostat ofknown mass to the corner.

-   -   NOTE: If the FD developed a channel leak during the performance        of the EVPA Assay, test the adherence on the opposite of the        haemostatic dressing to obtain a more accurate assessment of the        overall adherence.

2. Gently let go of the hemostat, taking care not to allow the hemostatto drop or twist. Turn the syringe so that the hemostat is near the topand allow the hemostat to peel back the dressing as far as the dressingwill permit. This usually occurs within 10 seconds. After the hemostathas stopped peeling back the dressing, rate the adherence of the bandageaccording to the following scale:

TABLE 1.1 Dressing Performance Score Amount of Adherence 4  90+% 375-90% 2 50-75% 1  ~50% 0.5 Only the plug holds the hemostat 0 Noadherence

Exclusion Criteria

The mesh support material must remain over the hole in the artery. If ithas shifted during the polymerization and does not completely cover thehole the haemostatic dressing must be excluded.

Success Criteria

Dressings that are given an adherence score of 3 are considered to havepassed the assay.

Failure Criteria

If a dressing does not adhere to the artery after application and/orprior to performing the EVPA assay, it is given a score of 0 and failsthe adherence test. If a dressing receives a score ≦2, the dressing isconsidered to have failed the Adherence Assay.

Weight Held Performance Assay

After the initial scoring of the “Adherence Test”, weights may then beadded to the hemostat in an incremental manner until the mesh supportmaterial is pulled entirely off of the artery. The maximum weight thatthe dressing holds is then recorded as a measure of the amount of weightthe dressing could hold attached to the artery.

Example 2

Similar to the need to evaluate a test article in the context of sealingand injury deep within surrounding tissue, there was also a need to testproducts that can seal injured tissue where the injured vessels aresmaller and thinner-walled than an aorta. The following assayaccomplishes this goal.

According to this modification, the porcine carotid artery is attachedto a barbed female connector using cotton thread with the connectivetissue side exposed. This is in contrast to the standard EVPA where theinternal side is exposed. As the carotid arteries used in the VA modelare more elastic and friable than the aorta, it is more difficult totreat or abrade the surface without damaging and compromising theartery. To ensure that no tears have occurred during the removal of thebulk of the connective tissue, the artery is connected to the barbedconnector and solution is pumped into it. If the artery is intact, a 1.5mm hole is punched into the artery using a biopsy punch.

After the artery is prepped, it is connected to the pump system andplaced on top of a piece of foam with a concave “hollow” cut into thesurface. This serves as a support for the artery during application ofthe FD and “compression” of the artery. The test article is applied tothe top of the hole and wet with 37° C. 0.9% NaCl. The artery is coveredwith plastic wrap, and a weight warmed to ˜38-40° C. is then placed ontop of the artery. The artery is partially compressed instead of beingpressed flat because of the support of the foam.

After the weight has been applied for 5 min., it is then removed, andthe pump is turned on. When the solution is coming out of the end of theartery, it is then clamped and allowed to pressurize until 250 mmHg or aleak occurs, whichever comes first.

In development of the assay, the following variables were considered andtested:

Tissue Selection: In order to mimic a vascular access procedure, atissue substrate that was elastic yet strong was needed. Contact withrendering companies such as PelFreeze and Animal Technologies revealed 2types of arteries collected that could be potentially used to mimic thevascular access procedure: porcine renal arteries and porcine carotidarteries. These arteries were comparable in size to a human femoralartery. Both types were purchased to examine their usefulness. Theporcine renal artery was too short in useable length (less than 2″), tosmall an internal diameter, and not as elastic as desired. The porcinecarotid artery, however, was highly elastic and offered useable segmentsof 3-5″ without branching or collateral arteries.

Artery Hole Size: To determine a size to use for the assay, the actualsurgical procedure was mimicked insofar as possible. A hole was put intothe artery using an 18-gauge needle. A 200 uL pipette tip was thenpushed into the hole to the point where the diameter was ˜3.5 mm, justlarger than a 10F catheter. The tip was left in place for 2 hrs. and wasthen removed. The resulting hole was larger than the initial 18-gaugeneedle punch and, when compared to 2.8, 2.0, and 1.5 mm holes, was verysimilar to the 1.5 mm hole produced by the biopsy punch.

Surface Preparation: In the EVPA assay, the interior surface of aporcine aorta is gently abraded using the edge of a pair of scissors toprovide a “damaged” surface to which the FD would adhere, mimickinglarge trauma. For the vascular access procedure, obtaining a uniform,reproducible surface on which to test the FD was important. Startingwith the familiar, the carotid artery was turned inside-out and abraded.However, this did not work as the carotid artery is highly elastic, andthe scraping of the surface created tears that rendered the arteryunusable. Using the exterior surface, the arteries that had theconnective tissue carefully removed down to the level of the arteryprovided a surface that was uniform and best mimicked the vascularaccess procedure.

Integration of the Artery into the Pump System: To best mimic thevascular access procedure, the use of the artery without any internalsupport to interfere with compression was desired. In order toincorporate the artery into the pump system, it was necessary to attachthe artery at one end to the tubing and still have an open end to allowsolution flow prior to pressurization. After examining different typesof tubing and connectors, a barbed low-pressure female connector waschosen. The barb could be either ⅛″ or ¼″, depending upon the innerdiameter of the carotid artery. To attach the artery to the barbed end,cable ties, o-rings, and thread were tested. Only the thread preventedleakage during pressurization.

Arterial Support: In trying to partially-compress the artery on a flatsurface, it became clear that some form of support was needed to preventthe artery from shifting during application of the FD and to preventtotal compression of the artery. A variety of materials were tested,including gel packs, Styrofoam packaging material, and foam pieces. Foampieces that had a concave trough cut into the top surface offered thebest support: the trough held the artery in place, and it was cut justdeep enough to allow partial compression of the artery.

Compression Method: In the actual surgical procedure, hemostasis is morecommonly achieved by manual compression of the artery for a period of˜20 min. During this time, arterial flow is maintained. Application of aweight to the artery was tested in order to mimic this at the lab bench.Various weights in beakers just large enough to contain the weight weretested on arteries in the foam arterial support. With this set-up, both200 g and 500 g weight inside a glass beaker (to provide a uniformsurface for compression) just large enough to accommodate the weightproved to be ideal for compression. Weights lower or higher providedinsufficient or too much compression, respectively.

Temperature maintenance: FXIII, a component of the FD that isresponsible for cross-linking of fibrin monomers, is thermally labile,and the assay needs maintained around normal body and wound temperaturesof 34-36° C. As this set-up cannot be easily transferred to an incubatoras in the EVPA, another method had to be devised. Various methods wereconsidered such as warmed gel packs, heating pads, and warming lamps.While these methods would produce a warmer-than-ambient temperature,they were difficult to control to the level that this assay requires.The most practical method was the use of a heat block set to 37° C.While a heat block can maintain a constant temperature for very longperiods of time, they were not sufficient to warm the artery and FD to34-36° in the 5 minute time frame of the assay. As the weight that isapplied could be a potential heat source, it was warmed in the incubatorprior to application, and this addition to the 37° C. heat block wassufficient to maintain the 34-36° C. temperature range.

Data Collection: For this assay, the following pieces of data arecollected: amount of saline required to wet the dressing, ease ofwetting, artery temperature after the incubation period, maximumpressure obtained, failure mode (channel leak, leak through plug),qualitative assessment of the adherence of the dressing to the artery,and overall comments on dressing appearance (mottled, pre-formed fibrin,thin, etc.)

1 Test Protocol for Ex Vivo Porcine Carotid Artery Assay (EVPCA)

Equipment and Supplies

In-line high pressure transducer (Ashcroft Duralife or equivalent)

Peristaltic pump (Pharmacia Biotech, Model P-1 or equivalent)

Voltmeter (Craftsman Professional Model 82324 or equivalent)

Computer equipped with software for recording pressure or voltageinformation

Tygon tubing (asst. sizes) with attachments

Water bath (Baxter Durabath or equivalent), preset to 37° C.

Heat Block (Thermolyne Type 16500 Dri-Bath or equivalent)

Incubation chamber (VWR, Model 1400G or equivalent), preset to −40° C.

Thermometer to monitor temperatures of water bath, heat block, and oven

Calibration weights: 200 g and 500 g

Beakers to hold calibration weights

Biopsy punch(es), 1.5 mm or other required sizes

Assorted forceps, hemostats, and scissors

P-200 and P-100 Pipetman with tips

Plastic ⅛″ and ¼″ low pressure fittings, female connector with a barbedtubing connection

Plastic strips ⅛″ and ¼″ wide

Materials and Chemicals

Porcine carotid arteries (xxx or equivalent)

0.9% Saline, maintained at 37° C.

Red food coloring

Quilting thread or other heavy-gauge thread

Plastic Wrap

Foam pieces with a concave area cut into the surface

Preliminary Procedures

Artery Cleaning and Storage

1. Store arteries at −20° C. until used.

2. Thaw arteries at 37° C. in HP bath.

3. Clean fat and connective tissue from exterior surface of artery.

4. The arteries may be refrozen to −20° C. and stored until use.

Artery Preparation for Assay

1. Make sure that all connective tissue is removed from the artery.

2. If any collateral arteries or other large holes are visible, cut theartery at the hole. If a small section of artery is produced from thecut, discard it. If 2 pieces are produced that are at least 1½″ long,both pieces may be used for assays.

3. Insert the barbed end of a low pressure connector, either ⅛″ or ¼″depending upon the internal diameter of the artery, into the larger endof the artery.

4. Cut a piece of quilting or other heavy thread ˜6″ long. Using asquare knot, tie the artery to the connector so that it does not comeoff of the connector.

Note: As it is possible to tear the artery during the cleaning process,it is important to “leak test” the artery prior to performing the assay.

5. Connect the artery to the male connector at the end of the tubingattached to the pump system.

6. Turn on the pump with the open end of the artery pointed upwards andallow the artery to fill with the red 0.9% NaCl. When the artery isfull, clamp the artery closed using a hemostat.

7. Watch the artery as it pressurizes to see if any holes or tears arepresent. If a hole is present, turn off the pump, unclamp the arteryover a beaker to catch the saline solution, and disconnect it from thepump system.

For arteries with holes

-   -   If the hole is near the open end of the artery, cut off the        artery at the hole, leaving the artery attached to the        connector.    -   If the hole is near the connector, remove the artery from the        connector, cut the artery at the hole, and re-attach it to the        connector as outlined above.    -   For arteries that have pieces cut off, the remaining piece        should be at least 1½″ long. If not, it should be discarded.    -   If the hole is near the middle of the artery, check the size of        the hole. If it is less than 1.5 mm, it may be used for the        assay as a hole may be punched around it. If the hole is larger        than 1.5 mm, the artery should be discarded.

For arteries without holes

-   -   If no holes are seen, allow the artery to pressurize to ˜100        mmHg (a reading of 2.0 on the pressure gauge). Turn off the pump        and unclamp the artery over a beaker, and disconnect it from the        pump system.    -   If any holes become visible during this period, unclamp the        artery over a beaker, disconnect form the pump system, and fix        the artery according to the procedures outlined above.        Note: After the artery has been inspected and any unwanted holes        addressed, the test hole may then be punched in the artery    -   8. Insert a plastic strip into the open end of the artery so        that it goes most of the way into the artery.    -   9. Using the biopsy punch, carefully punch a hole in the artery.        Make sure that the punch connects with the plastic strip so that        no additional holes are punched in the artery.    -   10. The punch should totally remove the center portion. If it        does not, gently remove it with forceps or by re-cutting it        using the biopsy punch.    -   11. Place the artery in the warmed, moistened container and        place in the ˜40° C. incubation chamber to keep the artery moist        prior to assay

Solution and Equipment Preparation

1. Turn on the heat block and check to see that it is maintained at 37°C.2. Check to see that the water bath is maintained at 37° C. andincubation chamber is maintained at ˜40° C.3. Make sure that there is sufficient 0.9% saline in the pump'sreservoir for completion of the day's assays. Add more if needed.4. Place 0.9% saline into containers in a 37° C. water bath so that thesolutions will be warmed prior to performing the assay.5. The peristaltic pump should be calibrated so that it deliversapproximately 3 ml/min. If not, adjust the settings at this point.6. Check the tubing for air bubbles. If bubbles exist, turn on the pumpand allow the 0.9% saline to flow until all bubbles are removed.

Application of the FD or HD

1. Place a piece of foam with the concave surface on top of the heatingblock and cover with a piece of plastic wrap.2. Remove an artery from the warming box and attach it to the pumpsystem.3. Allow the artery to rest in the concave hollow of the foam piece.4. Open the haemostatic dressing pouch and remove haemostaticdressing(s). Place any extras in the vacuum dessicator.5. Place the dressing, mesh support material side UP (or the sideclosest to the bottom of the mold if no support material is present),over the hole in the artery6. Slowly wet the haemostatic dressing with an amount of salineappropriate for the article being tested

-   -   NOTE: A standard (13-15 mg/cm² of fibrinogen) 2.4×2.4 cm        haemostatic dressing should be wet with 800 μl of saline or        other blood substitute. A dressing of 1.5×1.5 cm would require        300 μl of saline or other blood substitute, and a 0.7×0.7 cm        dressing would require 70 μl of saline or other blood        substitute. The amount of saline used can be adjusted depending        on the requirements of the particular experiment being        performed; however, any changes should be noted on the data        collection forms.    -   NOTE: Wet the haemostatic dressing drop wise with 0.9% saline        warmed to 37° C. or other blood substitute, taking care to keep        the saline from running off the edges. Any obvious differences        in wetting characteristics from the positive control should be        noted on data collection forms.        7. Cover the artery with plastic wrap, taking care that the        dressing doesn't slide around on the surface of the artery.        8. Place a warmed weight carefully on top of the dressing so        that it does not shift off of the hole in the artery.        9. Allow the weight to remain on the artery on top of the 37° C.        heat block for the duration of the polymerization time.    -   NOTE: Time, pressure, and hole size can be altered according to        the requirements of the experiment; changes from the standard        conditions should be noted on the data collection forms        10. After polymerization, carefully unwrap the artery and note        the condition of the haemostatic dressing. Any variation from        the positive control should be noted on the data collection        form.

EXCLUSION CRITERION: The mesh support material must remain over the holein the artery. If it has shifted during the polymerization and does notcompletely cover the hole the haemostatic dressing must be excluded.

Testing Procedure

A diagram of testing equipment set-up is shown in FIG. 1.

TABLE 2.1 Conversion Table for Pressure (PSI) to mmHg and VoltagePressure Gauge mm Hg Voltage Reading (PSI) Equivalent Equivalent 1.0 501.25 2.0 100 1.50 3.0 150 1.75 4.0 200 2.00 5.0 250 2.25

Equipment and Artery Assembly

-   -   1. After the polymerization period is complete, carefully remove        the plastic wrap so that the dressing is not disturbed.    -   2. Turn on the pump and gently lift the open end of the artery        with a hemostat. Allow the artery to fill to the top with 0.9%        NaCl. This is done to minimize air bubbles in the system.    -   3. The system should be operated according to a pre-determined        range of pressures and hold times as appropriate for the article        being tested. Should the pressure drop below the desired maximum        during the hold period, the pump should be turned on again until        the maximum pressure is achieved.    -   4. Should a leak in the system develop other than failure of the        FD or HD (i.e. leaking from a hole in the artery, etc.),        attempts to correct the problem should be taken. This might        involve clamping the leak for the remainder of the assay. Should        the attempts to fix the problem be ineffective, the test article        will be excluded from analysis and called a “system failure”        (See Exclusion Criteria below).    -   5. Following the conclusion of testing, the haemostatic dressing        is subjectively assessed with regard to adhesion to the artery        and formation of a plug in the artery hole. Any variations from        the positive control should be noted on the data collection        form.

Success/Fail and Exclusion Criteria

Success Criteria

-   -   1. Haemostatic dressings that are able to withstand various        pressures for 3 minutes are considered to have passed the assay.    -   2. When a haemostatic dressing has successfully passed the assay        the data collection should be stopped immediately so that the        natural decrease in pressure that occurs in the artery once the        test is ended isn't included on the graphs. Should the operator        fail to stop data collection, these points can be deleted from        the data file to avoid confusing the natural pressure decay that        occurs post-test with an actual dressing failure.    -   3. The entire testing period from application of the haemostatic        dressing to completion must fall within pre-established        criteria.        -   NOTE: For a single-step increase to maximum pressure the            entire testing period should not exceed 15 minutes. Other            time limits may be established for other test procedures,            and should be noted on the data collection forms.    -   4. The maximum pressure reached should be recorded on the data        collection form.        -   NOTE: Typical challenge is 250 mmHg for three minutes in one            step, but that may be altered based on the article being            tested. The pressure, for example, may be increased in            “steps” with holds at various pressures until the 250 mmHg            is achieved. One example is increasing the pressure in 50            mmHg increments with a 1 minute hold at each step to ensure            that the FD or HD can hold these pressures.

Failure Criteria

-   -   1. Haemostatic dressings that start leaking saline at the point        of FD or HD attachment at any point during testing are        considered to have failed the assay.        -   NOTE: Build failures that are caused by artery swelling can            be ignored and the test continued or re-started (as long as            the total testing time doesn't fall beyond the established            limit).    -   2. When leakage from the FD or HD does occur, the pressure        should be allowed to fall ˜20 mmHg before data collection is        stopped so that the failure is easily observed on the graphs.    -   3. The pressures at which leakage occurred should be recorded on        the data collection form.    -   4. Should the data collection stop in the middle of the        experiment due to equipment failure the data can be collected by        hand at 5 second intervals until the end of the test or        haemostatic dressing failure, whichever happens first. The data        points should be recorded on the back of the data collection        form, clearly labeled, and entered by hand into the data tables.

Exclusion Criteria

-   -   1. If the total testing period exceeds the maximum allowed for        that procedure, regardless of cause, results must be excluded.    -   2. If there are leaks from holes that can't be fixed by clamping        or finger pressure the results must be excluded.    -   3. If the mesh support material does not completely cover the        hole in the artery, the results must be excluded

Example 3

For all dressings, ERL fibrinogen lot 3130 was formulated in CFB. Thefinal pH of the fibrinogen was 7.4±0.1. The fibrinogen concentration wasadjusted to 37.5 mg/ml. Once prepared the fibrinogen was placed on iceuntil use. Thrombin was formulated in CTB. The final pH of the thrombinwas 7.4±0.1. The thrombin was adjusted to deliver 0.1 units/mg ofFibrinogen or 25 Units/ml thrombin. For the group with shredded supportmaterial dispersed within, it was cut into approximately 1 mm×1 mmpieces and dispersed within the thrombin solution prior to filling themolds. Once prepared the thrombin was placed on ice until use. Thetemperature of the fibrinogen and thrombin prior to dispensing was 4°C.±2° C. Cylindrical molds made of 10 or 3 mL polypropylene syringes(Becton Dickinson) with the luer-lock end removed were used. Theplungers were withdrawn to the 6 mL and 2 mL mark respectively. Fordressings utilizing a support material, the support material was cut andplaced into each mold and pushed down until it was adjacent to theplunger. Once prepared the molds were placed upright and surrounded bydry ice, leaving the opening exposed at the top. 1 ml of fibrinogen and0.15 mL of thrombin (with or without support material dispersed within)were dispensed into the 10 mL molds and 1 ml of fibrinogen and 0.15 mLof thrombin (with or without support material dispersed within) weredispensed into the 3 mL molds, which were allowed to freeze for 5minutes. The molds were then placed into the −80° C. freezer for atleast two hours before being placed into the freeze dryer andlyophylized as described above. The compositions are shown in Table 3.1below.

TABLE 3.1 Mold Fibrinogen Dose Thrombin Dose T:F Size (mg/cm²) (U/cm²)Ratio  3 ml 75 7.5 0.1 10 ml 25 2.5 0.1

Upon removal from the lyophylizer, both groups were performance testedin a modified EVPA assay as described in Example 1 above. Briefly, aplastic foam form was slipped over the artery. This covering had a holein it that corresponded to the hole in the artery and the surroundingtissue. Warm saline was added to the surface of the dressing and themold was immediately passed down thru the hole in the foam to the arterysurface. The plunger was then depressed and held by hand for 3 minutes,after which the mold was withdrawn as the plunger was depressed further.At this point the artery was pressurized and the assay continued asdescribed in Example 1 above.

Results

TABLE 3.1 Mold EVPA Result Maximum Support Material Size (@250 mmHg)Pressure None 10 ml Pass >250 mmHg    Dexon Mesh Backing 10 ml Pass ″ ″ 3 ml Pass ″ Shredded Dexon Mesh 10 ml Pass ″ (Dispersed) Shredded DexonMesh  3 ml Fail 150 mm Hg (Dispersed)

Conclusions: Dressings that included no support material or a DEXON™mesh support material performed well, with all passing the EVPA test at250 mmHg. When the support material was dispersed throughout thecomposition, the dressings also performed well, with the large size (10mL mold) dressings holding the full 250 mmHg of pressure, while thesmaller held up to 150 mmHg of pressure. This indicates that the use ofa support material may be optional, and it's location may be on the‘back’ of the dressing, or dispersed throughout the composition, asdesired.

The results demonstrate that the dressings were effective at the highestpressure tested regardless of size, and that they functioned effectivelyregardless of the presence or absence of the support material. Higherperformance was associated with the presence of support material, and alarger applicator.

Example 4

Dexon™ Mesh support material was cut to fit into and placed into eachPETG 1.5×1.5 cm mold. Fifteen microliters of 2% sucrose was pipeted ontop of each of the four corners of the support material and the moldswere placed inside a −80° C. freezer. Once completed the molds wereplaced in a −80° C. freezer. All molds remained in the −80° C. freezerfor at least 60 minutes. Enzyme Research Laboratories (ERL) Fibrinogenlot 3150 was formulated in 100 mM Sodium Chloride, 1.1 mM CalciumChloride, 10 mM Tris, 10 mM Sodium Citrate, and 1.5% Sucrose (Fibrinogencomplete buffer). In addition, Human Serum Albumin was added to 80 mg/gof total protein and Tween 80 (non-animal source) was added to 15 mg/gtotal protein. The final pH of the fibrinogen was 7.4+/−0.1. Thefibrinogen concentration was adjusted to 37.5 mg/ml. Once prepared thefibrinogen was placed on ice until use. Thrombin was formulated in 150mM Sodium Chloride, 40 mM Calcium Chloride, 10 mM Tris and 100 mML-Lysine. The final pH of the thrombin was 7.4+/−0.1. The thrombin wasadjusted to 25 Units/ml thrombin, resulting in 0.1 units/mg ofFibrinogen or 1.3 U/cm'. Once prepared the thrombin was placed on iceuntil use. The temperature of the fibrinogen and thrombin prior todispensing was 4° C.+/−2° C. Molds were removed from the −80° C. freezerand placed on a copper plate that was placed on top of dry ice. A repeatpipetor was filled with fibrinogen and second repeat pipetor was filledwith thrombin. Simultaneously 0.8 ml of fibrinogen and 133 micro litersof thrombin were dispensed into each mold. Once the molds were filled,they were returned to the −80° C. freezer for at least two hours beforebeing placed into a pre-cooled Genesis™ lyophylizer (Virtis, Gardiner,N.Y.). The chamber was sealed and the temperature equilibrated. Thechamber was then evacuated and the dressings lyophilized as described inExample 3.

Test articles of a different size were also prepared as follows. Supportmaterial was cut and placed into each PETG 0.7×0.7 cm mold. Fivemicroliters of 2% sucrose was pipeted on top of each of the four cornersof the support material and the molds were placed inside a −80° C.freezer. Once completed the molds were placed in a −80° C. freezer. Allmolds remained in the −80° C. freezer for at least 60 minutes. EnzymeResearch Laboratories (ERL) Fibrinogen lot 3150 was formulated in 100 mMSodium Chloride, 1.1 mM Calcium Chloride, 10 mM Tris, 10 mM SodiumCitrate, and 1.5% Sucrose (Fibrinogen complete buffer). In addition,Human Serum Albumin was added to 80 mg/g of total protein and Tween 80(non-animal source) was added to 15 mg/g total protein. The final pH ofthe fibrinogen was 7.4+/−0.1. The fibrinogen concentration was adjustedto 39.2 mg/ml. Once prepared the fibrinogen was placed on ice until use.Thrombin was formulated in 150 mM Sodium Chloride, 40 mM CalciumChloride, 10 mM Tris and 100 mM L-Lysine. The final pH of the thrombinwas 7.4+/−0.1. The thrombin was adjusted to 25 Units/ml thrombin, whichresulted in a final composition of 0.1 units/mg of Fibrinogen or 1.3 Uthrombin/cm². Once prepared the thrombin was placed on ice until use.The temperature of the fibrinogen and thrombin prior to dispensing was4° C.+/−2° C. Molds were removed from the −80° C. freezer and placed ona copper plate that was placed on top of dry ice. A repeat pipetor wasfilled with fibrinogen and second repeat pipetor was filled withthrombin. Simultaneously 0.17 ml of fibrinogen and 26 micro liters ofthrombin were dispensed into each mold. Once the molds were filled, theywere returned to the −80° C. freezer for at least two hours before beingplaced into the freeze dryer and lyophylized as described above.

The performance of the test articles was determined using the EVPCAassay as described in Example 2 above.

Results:

TABLE 4.1 Fibrinogen Thrombin % % % % Test Article Dose Dose T:FReaching Reaching Reaching Reaching Size (cm²) (mg/cm²) (U/cm²) (U/mg)100 mmHg 150 mmHg 200 mmHg 250 mmHg 0.7 13 1.3 0.1 100 80 40 40 1.5 131.3 0.1 100 80 80 60

Example 5

Dexon™ Mesh support material was cut to fit into and placed into eachPETG 1.5×1.5 cm mold. Fifteen microliters of 2% sucrose was pipeted ontop of each of the four corners of the support material and the moldswere placed inside a −80° C. freezer. PETG 1.5×1.5 cm molds that did notcontain support material were also placed inside the −80° C. freezer. Ina third group, the same amount of support material was cut into smallpieces (approximately less than 2 mm.×2 mm) and placed into PETG 1.5×1.5cm molds(these dressings are referred to as having their supportmaterial ‘dispersed’). Once completed the molds were placed in a −80° C.freezer. All molds remained in the −80° C. freezer for at least 60minutes. Enzyme Research Laboratories (ERL) Fibrinogen lot 3130 wasformulated in 100 mM Sodium Chloride, 1.1 mM Calcium Chloride, 10 mMTris, 10 mM Sodium Citrate, and 1.5% Sucrose (Fibrinogen completebuffer). In addition, Human Serum Albumin was added to 80 mg/g of totalprotein and Tween 80 (non-animal source) was added to 15 mg/g totalprotein. The final pH of the fibrinogen was 7.4+/−0.1. The fibrinogenconcentration was adjusted to 36.56 mg/ml and 14.06 mg/ml. Once preparedthe fibrinogen was placed on ice until use. Thrombin was formulated in150 mM Sodium Chloride, 40 mM Calcium Chloride, 10 mM Tris and 100 mML-Lysine. The final pH of the thrombin was 7.4+/−0.1. The thrombin wasadjusted to deliver 0.01, 0.1 or 1 units/mg of Fibrinogen or 2.5, 25 or250 Units/ml thrombin. Once prepared the thrombin was placed on iceuntil use. The temperature of the fibrinogen and thrombin prior todispensing was 4° C.+/−2° C. Molds were removed from the −80° C. freezerand placed on a copper plate that was placed on top of dry ice. A repeatpipetor was filled with fibrinogen and second repeat pipetor was filledwith thrombin. Simultaneously 0.8 ml of fibrinogen and 133 micro litersof thrombin were dispensed into each mold. Once the molds were filled,they were returned to the −80° C. freezer for at least two hours beforebeing placed into the freeze dryer. Table 5.1 shows the experimentaldesign.

TABLE 5.1 Experimental Design Fibrinogen Dose Thrombin Dose T:F Support(mg/cm²) (U/cm²) (U/mg) material 5 0.05 0.01 Yes 5 0.05 0.01 No 5 0.050.01 Dispersed 5 0.5 0.1 Yes 5 0.5 0.1 No 5 0.5 0.1 Dispersed 5 5 1 Yes5 5 1 No 5 5 1 Dispersed 13 0.13 0.01 Yes 13 0.13 0.01 No 13 0.13 0.01Dispersed 13 1.3 0.1 Yes 13 1.3 0.1 No 13 1.3 0.1 Dispersed 13 13 1 Yes13 13 1 No 13 13 1 Dispersed

The performance of the test articles was determined using the EVPCAassay as described in Example 2 above.

Results:

TABLE 5.2 Fibrinogen Thrombin Thrombin % % % Dose Dose (U/mg SupportReaching Reaching Reaching (mg/cm²) (U/cm²) fibrinogen) material 150mmHg 200 mmHg 250 mmHg 5 0.05 0.01 Yes 66.6 50 50 5 0.05 0.01 No 0 16 05 0.05 0.01 Dispersed 13.3 0 0 5 0.5 0.1 Yes 66.6 66 50 5 0.5 0.1 No 6020 0 5 0.5 0.1 Dispersed 40 0 0 5 5 1 Yes 33.3 0 0 5 5 1 No 0 0 0 5 5 1Dispersed 0 0 0 13 0.13 0.01 Yes 100 50 33.3 13 0.13 0.01 No 33.3 0 0 130.13 0.01 Dispersed 20 20 0 13 1.3 0.1 Yes 66.6 50 16.6 13 1.3 0.1 No 00 0 13 1.3 0.1 Dispersed 100 80 40 13 13 1 Yes 33.3 33.3 33.3 13 13 1 No33.3 0 0 13 13 1 Dispersed 33.3 16.6 16.6

Example 6

Dexon™ Mesh support material was cut to fit into and placed into eachPETG 0.7×0.7 cm mold. Five microliters of 2% sucrose was pipeted on topof each of the four corners of the support material and the molds wereplaced inside a −80° C. freezer. Once completed the molds were placed ina −80° C. freezer. All molds remained in the −80° C. freezer for atleast 60 minutes. Enzyme Research Laboratories (ERL) Fibrinogen lot 3130was formulated in 100 mM Sodium Chloride, 1.1 mM Calcium Chloride, 10 mMTris, 10 mM Sodium Citrate, and 1.5% Sucrose (Fibrinogen completebuffer). In addition, Human Serum Albumin was added to 80 mg/g of totalprotein and Tween 80 (non-animal source) was added to 15 mg/g totalprotein. The final pH of the fibrinogen was 7.4+/−0.1. The fibrinogenconcentration was adjusted to 39.2 mg/ml and 32.06 mg/ml. Once preparedthe fibrinogen was placed on ice until use. Thrombin was formulated in150 mM Sodium Chloride, 40 mM Calcium Chloride, 10 mM Tris and 100 mML-Lysine. The final pH of the thrombin was 7.4+/−0.1. The thrombin wasadjusted to deliver 1 unit/mg of Fibrinogen or 250 Units/ml thrombin.Once prepared the thrombin was placed on ice until use. The temperatureof the fibrinogen and thrombin prior to dispensing was 4° C.+/−2° C.Molds were removed from the −80° C. freezer and placed on a copper platethat was placed on top of dry ice. A repeat pipetor was filled withfibrinogen and second repeat pipetor was filled with thrombin.Simultaneously 0.2 ml of fibrinogen and 33 micro liters of thrombin weredispensed into each mold. Once the molds were filled, they were returnedto the −80° C. freezer for at least two hours before being placed intothe freeze dryer. Table 6.1 shows the experimental design.

TABLE 6.1 Experimental Design Fibrinogen Dose Thrombin Dose T:F Support(mg/cm²) (U/cm²) (U/mg) material 16 16.1 1 Yes 13 13.0 1 Yes

The performance of the test articles was determined using the EVPCAassay as described in Example 2 above.

Results:

TABLE 6.2 Fibrinogen % Pass % Pass % Pass Dose EVCPA at EVCPA at EVCPAat (mg/cm²) 150 mm Hg 200 mm Hg 250 mm Hg 16 66.6 33.3 16.6 13 0 0 0

Example 7

Dexon™ Mesh support material was cut to fit into and placed into eachPETG 1.5×1.5 cm mold. Fifteen microliters of 2% sucrose was pipetted ontop of each of the four corners of the support material and the moldswere placed inside a −80° C. freezer. Once completed the molds wereplaced in a −80° C. freezer. All molds remained in the −80° C. freezerfor at least 60 minutes. Enzyme Research Laboratories (ERL) Fibrinogenlot 3170P was formulated in 100 mM Sodium Chloride, 1.1 mM CalciumChloride, 10 mM Tris, 10 mM Sodium Citrate, and 1.5% Sucrose (Fibrinogencomplete buffer). In addition, Human Serum Albumin was added to 80 mg/gof total protein and Tween 80 (non-animal source) was added to 15 mg/gtotal protein. The final pH of the fibrinogen was 7.4+/−0.1. Thefibrinogen concentration was adjusted to 36.56 mg/ml and 14.06 mg/ml.Once prepared the fibrinogen was placed on ice until use. Thrombin wasformulated in 150 mM Sodium Chloride, 40 mM Calcium Chloride, 10 mM Trisand 100 mM L-Lysine. The final pH of the thrombin was 7.4+/−0.1. Thethrombin was adjusted to deliver 0.001, or 0.0001 units/mg of Fibrinogenor 0.25, 0.025 Units/ml thrombin. Once prepared the thrombin was placedon ice until use. The temperature of the fibrinogen and thrombin priorto dispensing was 4° C.+/−2° C. Molds were removed from the −80° C.freezer and placed on a copper plate that was placed on top of dry ice.A repeat pipetor was filled with fibrinogen and second repeat pipetorwas filled with thrombin. Simultaneously 0.8 ml of fibrinogen and 133micro liters of thrombin were dispensed into each mold. Once the moldswere filled, they were returned to the −80° C. freezer for at least twohours before being placed into the freeze dryer. Table 7.1 shows theexperimental design.

TABLE 7.1 Experimental Design Fibrinogen Dose Thrombin Dose T:F Support(mg/cm2) (U/cm²) (U/mg) material 13 0.013 0.001 Yes 13 0.0013 0.0001 Yes5 0.005 0.001 Yes 5 0.0005 0.0001 Yes

The performance of the test articles was determined using the EVPCAassay as described in Example 2 above.

Example 8

Previously manufactured lyophylized mixtures of fibrinogen & thrombin(lot # 012408) were placed into a grinder (Krups) and ground (5 seconds)into a powder. The powdered dressings were placed into a 50 ml conicalcentrifuge tube. Twenty-five grams of sucrose was ground into powder andplaced into another 50 ml conical centrifuge tube. Table 8.1 shows thedesign of the experiment.

TABLE 8.1 Experimental Design Fibrinogen Thrombin Support Weight ofWeight of Dose Dose T:F material/ Dressing Sucrose (mg/cm²) (U/cm²)(U/mg) Placement Powder (g) (g) 5 1.3 0.26 Yes 0.008 0.092 13 1.3 0.1Yes/Bottom 0.208 0.792 13 1.3 0.1 Yes/Top 0.208 0.792 26 1.3 0.05Yes/Bottom 0.416 0.584 26 1.3 0.05 Yes/Top 0.416 0.584 26 1.3 0.05Yes/Middle 0.416 0.584 56 1.3 0.023 Yes/Top 0.12 0.00 56 1.3 0.023Yes/Middle 0.12 0.00 450 1.3 0.003 Yes/Middle 0.95 0.00 5 1.3 0.26 No0.008 0.092 13 1.3 0.1 No 0.208 0.792 26 1.3 0.05 No 0.416 0.584 56 1.30.023 No 0.12 0.00 5 1.3 0.26 Dispersed 0.008 0.092 13 1.3 0.1 Dispersed0.208 0.792 26 1.3 0.05 Dispersed 0.416 0.584 56 1.3 0.023 Dispersed0.12 0.00

For each group 0.1 g of the powder/sucrose was weighed and placed into aCarver 13 mm Evacuable Pellet Die. For pellets that had a supportmaterial, 75 mg of the support material was placed in one of fourlocations. In the first, the support material (Dexon™ mesh) was placedinto the die, followed by the addition of the powder, these are referredto as being in the ‘bottom’ position. When the powder was placed intothe die followed by the support material (Dexon™ mesh) these arereferred to as in the ‘top’ position. For pellets with the supportmaterial in the ‘middle’ position, 50 mg of the powder/sucrose wasweighed and placed into a Carver 13 mm Evacuable Pellet Die, followed bythe support material (75 mg of Dexon™ mesh) which was then topped off byanother 50 mg of the powder/sucrose mixture. For pellets that haddispersed support material, the powder and 75 mg of shredded supportmaterial (Dexon™ mesh) were added to the die at the same time and mixedfor 5 seconds with a pipette tip. Once the die was filled with theappropriate material, it was placed in a Carver 4350 manual pelletpress. Pressure was applied to give an applied load of 1000 lbs. Theresulting pellets were removed and placed into a desiccator untiltested.

The performance of the test articles was determined using the EVPCAassay as described in Example 2 above. The test articles containing afibrinogen dose of 26 or 56 mg/cm² exhibited the best results.

Example 9

Previously manufactured lyophylized mixtures of fibrinogen & thrombinwere placed into a grinder (Krups) and ground (5 seconds) into a powder.The powdered dressings were placed into a 50 ml conical centrifuge tube.Twenty-five grams of sucrose was ground into powder and placed intoanother 50 ml conical centrifuge tube. Cylindrical molds made of 3 mLpolypropylene syringes (Becton Dickinson) with the luer-lock end removedwere used. The plungers were withdrawn to the 2 or 3 ml mark.

TABLE 9.1 Experimental Design Fibrinogen Thrombin Weight of Weight ofDose Dose T:F Support Dressing Sucrose (mg/cm²) (U/cm²) (U/mg) materialPowder (g) (g) 26 1.3 0.05 No .0208 0.0792 26 1.3 0.05 Yes .0208 0.079226 1.3 0.05 Dispersed .0208 0.0792 56 1.3 0.023 No 0.0448 0.0552 56 1.30.023 Yes 0.0448 0.0552 56 1.3 0.023 Dispersed 0.0448 0.0552 150 1.30.0087 No 0.12 0.0 150 1.3 0.0087 Yes 0.12 0.0 150 1.3 0.0087 Dispersed0.12 0.0 450 1.3 0.003 No 0.36 0.0 450 1.3 0.003 Yes 0.36 0.0 450 1.30.003 Dispersed 0.36 0.0

For dressings utilizing a support material, 75 mg of Dexon mesh supportmaterial was cut to fit into the mold and then placed into each mold andpushed down until it was adjacent to the plunger. Where syringes haddispersed support material, an equivalent amount of support material wasshredded and dispersed within the powder that was added to each syringe.For each group 0.1 g of the powder/sucrose was weighed and placed into aeach syringe, except for the 150 mg/cm² group which had 0.12 g added tothe syringe. Gelfoam™ was cut to fit into the mold and then placedinside the syringes, either alone or with 26 mg/cm² of dressing powder.

The performance of the test articles was determined using the EVPCAassay as described in Example 2 above. The results are shown in Table9.2 below.

Results:

TABLE 9.2 Fibrinogen % % % % % Dose Support Reaching Reaching ReachingReaching Reaching (mg/cm²) Material 60 mmHg 100 mmHg 150 mmHg 200 mmHg250 mmHg 26 Yes 100 100 0 0 0 26 Dispersed 0 0 0 0 0 26 No 0 0 0 0 0 26Gelfoam ™ 100 0 0 0 0 56 Yes 100 100 100 100 100 56 Dispersed 100 100100 100 100 56 No 0 0 0 0 0 150 Yes 100 100 100 100 50 150 Dispersed 100100 100 50 50 150 No 100 100 100 100 100 450 Yes 100 100 100 100 0 450Dispersed 100 100 100 100 100 450 No 100 100 100 0 0

Example 10

Enzyme Research Laboratories (ERL) Fibrinogen lot 3170P was formulatedin 100 mM Sodium Chloride, 1.1 mM Calcium Chloride, 10 mM Tris, 10 mMSodium Citrate, and 1.5% Sucrose (Fibrinogen complete buffer). Inaddition, Human Serum Albumin was added to 80 mg/g of total protein andTween 80 (non-animal source) was added to 15 mg/g total protein. Thefinal pH of the fibrinogen was 7.4+/−0.1. The fibrinogen concentrationwas adjusted to 37.5 mg/ml. Once prepared the fibrinogen was placed onice until use. Fibrinogen was diluted to 18.75 mg/ml and 9.4 mg/ml withFibrinogen complete buffer.

Enzyme Research Laboratories (ERL) Fibrinogen lot 3170P was formulatedin 100 mM Sodium Chloride, 1.1 mM Calcium Chloride, 10 mM Tris, 10 mMSodium Citrate, and 1.5% Sucrose (Fibrinogen complete buffer). Thisgroup did not contain Sucrose or Tween. The final pH of the fibrinogenwas 7.4+/−0.1. The fibrinogen concentration was adjusted to 37.5 mg/ml.Once prepared the fibrinogen was placed on ice until use. Thrombin wasformulated in 150 mM Sodium Chloride, 40 mM Calcium Chloride, 10 mM Trisand 100 mM L-Lysine. The final pH of the thrombin was 7.4+/−0.1. Thethrombin was adjusted to deliver 0.1 units/mg of Fibrinogen or 25Units/ml thrombin. Thrombin was diluted to 12.5 U/ml and 6.25 U/ml withThrombin buffer. Once prepared the thrombin was placed on ice until use.The temperature of the fibrinogen and thrombin prior to dispensing was4° C.+/−2° C. Microcentrifuge tubes (0.65 ml) were placed on dry ice.There were two groups of frozen plugs prepared with one frozen plug pergroup. One group did not have any support material, and the second groupcontained shredded support material (Dexon mesh) (0.1 g) dispersedwithin it. A repeat pipetor was filled with fibrinogen and second repeatpipetor was filled with thrombin. Simultaneously 0.5 ml of fibrinogenand 75 micro liters of thrombin were dispensed into each microcentrifugetube. Once each microcentrifuge tube was filled, they were transferredto a −80° C. freezer until tested. Table 10.1 shows the experimentaldesign.

TABLE 10.1 Experimental Design Fibrinogen Dose Thrombin Dose T:F Support(mg/Item) U/Item U/mg Material 18.75 1.875 0.1 Dispersed No Sucrose orTween 18.75 1.875 0.1 No No Sucrose or Tween 18.75 1.875 0.1 Dispersed18.75 1.875 0.1 No 9.4 0.94 0.1 Dispersed 9.4 0.94 0.1 No 4.7 0.47 0.1Dispersed 4.7 0.47 0.1 No

The performance of the test articles was determined using a modifiedEVPCA assay. The EVPCA assay (described in Example 2 above) was modifiedto further enhance the faithfulness of the assay to the actualconditions that may be encountered in vivo. As described in Example 3,the surrounding of the test blood vessel by closely fitting material canreplicate the use of these inventions in sealing an injury deep insidetissue. To further enhance this replication of such a clinical setting,tissue was substituted for the plastic foam that was wrapped around thevessel. The tissue may be chosen to best replicate the intendedanatomical location. In this Example commercial meat was used tosimulate the leg muscle of a patient undergoing a vascular accessprocedure. Sufficient tissue was used to simulate a depth of severalinches of muscle tissue. Other than this modification, and theemployment of an application device as described in Example #3, theassay was carried out as described in Example #2. The results are shownin Table 10.2 below.

Results:

TABLE 10.2 Fibrinogen Thrombin % % % % Dose Dose T:F Support ReachingReaching Reaching Reaching (mg/Item) (U/Item) U/mg Material 100 mmHg 150mmHg 200 mmHg 250 mmHg 18.75 No 1.875 0.1 Dispersed 50 50 50 50 Sucroseor Tween 18.75 No 1.875 0.1 No 50 0 0 0 Sucrose or Tween 18.75 1.875 0.1Dispersed 100 100 100 0 18.75 1.875 0.1 No 100 100 100 0 9.4 0.94 0.1Dispersed 100 0 0 0 9.4 0.94 0.1 No 0 0 0 0 4.7 0.47 0.1 Dispersed 0 0 00 4.7 0.47 0.1 No 0 0 0 0

Example 11

Previously manufactured lyophylized mixtures of fibrinogen & thrombin(lot # 012408) were placed into a grinder (Krups) and ground (5 seconds)into a powder. The powdered dressings were placed into a 50 ml conicalcentrifuge tube. Twenty-five grams of sucrose was ground into powder andplaced into another 50 ml conical centrifuge tube. Table 11.1 shows thedesign of the experiment.

TABLE 11.1 Experimental Design Weight of Fibrinogen Thrombin SupportDressing Dose Dose T:F material and Disk Powder Weight of (mg/cm²)(U/cm²) (U/mg) placement Modification (g) Sucrose (g) 26 1.3 0.05Yes/Dispersed Hole in 0.416 0.584 center of Disk 26 1.3 0.05Yes/Dispersed 12.5% 0.416 0.584 Removed as a pie shaped wedge

For each group 0.1 g of the powder/sucrose was weighed and placed into aCarver 13 mm. Evacuable Pellet Die. Seventy-five mg of shredded supportmaterial (Dexon™ mesh) was added to the powder and mixed for 5 secondswith a pipette tip. Once the die was filled with the appropriatematerial, it was placed in a Carver 4350 manual pellet press. Pressurewas applied to give an applied load of 1000 lbs. Once the pellets wereremoved from the die a small hole was placed in the center of two ofpellets using a 1/64″ drill bit. The other two pellets had ⅛″ of thepellet removed in a wedge-shaped piece with the vertex at the center ofthe pellet. The resulting pellets were removed and placed into adesiccator until tested.

The performance of the test articles was determined using the EVPCAassay as described in Example 2 above. With a modification that a 22gauge wire was placed into the artery hole and the test article was sliddown the wire to come in contact with the artery hole. Once the testarticle was delivered to the hole the wire was removed and the testproceeded as described. The results are shown in Table 11.2 below.

Results:

TABLE 11.2 Fibrinogen Support % % % % Dose Material Disk ReachingReaching Reaching Reaching (mg/cm²) Placement Modification 100 mmHg 150mmHg 200 mmHg 250 mmHg 26 Dispersed Hole in 50 50 50 50 center of Disk26 Dispersed 12.5% 50 0 0 0 Removed as a pie shaped wedge

Example 12

Enzyme Research Laboratories (ERL) Fibrinogen lot 3170P was formulatedin 100 mM Sodium Chloride, 1.1 mM Calcium Chloride, 10 mM Tris, 10 mMSodium Citrate, and 1.5% Sucrose (Fibrinogen complete buffer). Inaddition, Human Serum Albumin was added to 80 mg/g of total protein andTween 80 (non-animal source) was added to 15 mg/g total protein. Thefinal pH of the fibrinogen was 7.4+/−0.1. The fibrinogen concentrationwas adjusted to 37.5 mg/ml. Once prepared the fibrinogen was placed onice until use. Thrombin was formulated in 150 mM Sodium Chloride, 40 mMCalcium Chloride, 10 mM Tris and 100 mM L-Lysine. The final pH of thethrombin was 7.4+/−0.1. The thrombin was adjusted to deliver 0.1units/mg of Fibrinogen or 25 Units/ml thrombin. Once prepared thethrombin was placed on ice until use. The temperature of the fibrinogenand thrombin prior to dispensing was 4° C.+/−2° C.

Cylindrical molds made of 3 mL polypropylene syringes (Becton Dickinson)with the luer-lock end removed were used. The plungers were withdrawn tothe 1.0 ml mark.

Cylindrical molds were placed on dry ice. There were two groups ofcylindrical molds prepared with one cylindrical mold per group. Onegroup did not have any support material, and the second group containedshredded support material (0.1 gm Dexon™ mesh) dispersed within it. Arepeat pipetor was filled with fibrinogen and second repeat pipetor wasfilled with thrombin. Simultaneously 0.5 ml of fibrinogen and 75 microliters of thrombin were dispensed into each cylindrical mold. Once eachcylindrical mold was filled, they were transferred to a −80° C. freezeruntil tested. Table 12.1 shows the experimental design.

TABLE 12.1 Fibrinogen Thrombin T:F Support Dose (mg) (Units) (U/mg)Material 18.75 1.875 0.1 Dispersed 18.75 1.875 0.1 No

The performance of the test articles was determined using a modifiedEVPCA assay. The EVPCA assay (described in Example 2 above) was modifiedto further enhance the faithfulness of the assay to the actualconditions that may be encountered in vivo. As described in Example 3,the surrounding of the test blood vessel by closely fitting material canreplicate the use of these inventions in sealing an injury deep insidetissue. To further enhance this replication of such a clinical setting,tissue was substituted for the plastic foam that was wrapped around thevessel. The tissue may be chosen to best replicate the intendedanatomical location. In this Example commercial meat was used tosimulate the leg muscle of a patient undergoing a vascular accessprocedure. Sufficient tissue was used to simulate a depth of severalinches of muscle tissue. Other than this modification, and theemployment of an application device as described in Example #3, theassay was carried out as described in Example #2. The results are shownin table 12.2

Results:

TABLE 12.2 Fibrinogen % % % % Dose Thrombin T:F Support ReachingReaching Reaching Reaching (mg) (Units) U/mg Material 100 mmHg 150 mmHg200 mmHg 250 mmHg 18.75 1.875 0.1 Dispersed 100 100 50 0 18.75 1.875 0.1No 0 0 0 0

1. A device for treating wounded internal tissue in a mammal comprising:(a) an applicator; and (b) a haemostatically effective amount of asubstantially homogeneous haemostatic material consisting essentially ofa fibrinogen component and a fibrinogen activator.
 2. The device ofclaim 1, wherein said applicator comprises a plunger or tamper.
 3. Thedevice of claim 1, wherein said applicator comprises a guide wire. 4.The device of claim 1, wherein said haemostatic material includes atleast one support layer.
 5. The device of claim 4, wherein said supportlayer comprises a backing material.
 6. The device of claim 4, whereinsaid support layer comprises an internal support material.
 7. The deviceof claim 4, wherein said support layer comprises a resorbable material.8. The device of claim 4, wherein said support layer comprises anon-resorbable material.
 9. The device of claim 8, wherein saidnon-resorbable material is selected from the group consisting ofsilicone polymers, paper, gauze, plastics, non-resorbable suturematerials, latexes and suitable derivatives of thereof.
 10. The deviceof claim 4, further comprising at least one physiologically acceptableadhesive between said haemostatic material and said support layer. 11.The device of claim 7, wherein said resorbable material is selected fromthe group consisting of proteinaceous materials, carbohydrate substancesand resorbable suture materials.
 12. The device of claim 11, whereinsaid proteinaceous material is at least one substance selected from thegroup consisting of keratin, silk, fibrin, collagen, gelatin andsuitable derivatives thereof.
 13. The device of claim 11, wherein saidcarbohydrate substance is selected from the group consisting of alginicacid and salts thereof, chitin, chitosan, cellulose, n-acetylglucosamine, proteoglycans, glycolic acid polymers, lactic acidpolymers, glycolic acid/lactic acid co-polymers, suitable derivativesthereof and mixtures of two or more thereof.
 14. The device of claim 1,wherein said haemostatic material also contains a fibrin cross-linkerand/or a source of calcium ions.
 15. The device of claim 1, wherein saidhaemostatic material also contains one or more of the following: atleast one filler; at least one solubilizing agent; at least one foamingagent; and at least one release agent.
 16. The device of claim 15,wherein said filler is selected from the group consisting of sucrose,lactose, maltose, keratin, silk, fibrin, collagen, gelatin, albumin,polysorbate, chitin, chitosan., alginic acid and salts thereof,cellulose, proteoglycans, glycolic acid polymers, lactic acid polymers,glycolic acid/lactic acid co-polymers, and mixtures of two or morethereof.
 17. The device of claim 15, wherein said solubilizing agent isselected from the group consisting of sucrose, lactose, maltose,dextrose, mannose, trehalose, mannitol, sorbitol, albumin, sorbate,polysorbate, and mixtures of two or more thereof.
 18. The device ofclaim 15, wherein said release agent is selected from the groupconsisting of gelatin, mannitol, sorbitol, polysorbate, sorbitan,lactose, maltose, trehalose, sorbate, glucose and mixtures of two ormore thereof.
 19. The device of claim 15, wherein said foaming agent isselected from the group consisting of mixtures of sodiumbicarbonate/citric acid, sodium bicarbonate/acetic acid, calciumcarbonate/citric acid and calcium carbonate/acetic acid.
 20. The deviceof claim 1, wherein said haemostatic material also contains at least onetherapeutic supplement selected from the group consisting ofantibiotics, anticoagulants, steroids, cardiovascular drugs, growthfactors, antibodies (poly and mono), chemoattractants, anesthetics,antiproliferatives/antitumor agents, antivirals, cytokines, colonystimulating factors, antifungals, antiparasitics, antiinflammatories,antiseptics, hormones, vitamins, glycoproteins, fibronectin, peptides,proteins, carbohydrates, proteoglycans, antiangiogenins, antigens,nucleotides, lipids, liposomes, fibrinolysis inhibitors, procoagulants,anticoagulants, vascular constrictors and gene therapy reagents.
 21. Thedevice of claim 20, wherein said therapeutic supplement is present in anamount equal to or greater than its solubility limit in fibrin.
 22. Thedevice of claim 1, wherein said haemostatic material further contains atleast one binding agent in an amount effective to retain the physicalintegrity of said haemostatic material.
 23. The device of claim 22,wherein said binding agent is selected from the group consisting ofsucrose, mannitol, sorbitol, gelatin, maltose, povidone, chitosan,carboxymethylcellulose and derivatives thereof.
 24. The device of claim1, wherein said haemostatic material has been subjected to at least oneprocess selected from the group consisting of lyophilization, drying,spray-drying, vacuum drying and vitrification, and combinations of twoor more thereof.
 25. The device of claim 1, wherein said haemostaticmaterial has moisture content of at least 6%.
 26. The device of claim 1,wherein said haemostatic material has moisture content of less than 6%.27. The device of claim 1, wherein said haemostatic material is frozen.28. The device of claim 1, wherein said fibrinogen component is amammalian fibrinogen.
 29. The device of claim 28, wherein said mammalianfibrinogen is selected from the group consisting of bovine fibrinogen,porcine fibrinogen, ovine fibrinogen, equine fibrinogen, caprinefibrinogen, feline fibrinogen, canine fibrinogen, marine fibrinogen andhuman fibrinogen.
 30. The device of claim 1, wherein said fibrinogencomponent is selected from the group consisting of bird fibrinogen andfish fibrinogen.
 31. The device of claim 1, wherein said fibrinogencomponent is selected from the group consisting of human fibrinogen,human fibrin I, human fibrin II, human fibrinogen a chain, humanfibrinogen β chain, human fibrinogen γ chain, and mixtures of two ormore thereof.
 32. The device of claim 28, wherein said mammalianfibrinogen is selected from the group consisting of recombinantlyproduced fibrinogen and transgenic fibrinogen.
 33. The device of claim1, wherein said fibrinogen activator is selected from the groupconsisting of thrombins, prothrombins, snake venoms, and mixtures of anytwo or more thereof.
 34. The device of claim 33, wherein said thrombinis mammalian thrombin.
 35. The device of claim 34, wherein saidmammalian thrombin is selected from the group consisting of bovinethrombin, porcine thrombin, ovine thrombin, equine thrombin, caprinethrombin, feline thrombin, canine thrombin, murine thrombin and humanthrombin.
 36. The device of claim 33, wherein said thrombin is selectedfrom the group consisting of bird thrombin and fish thrombin.
 37. Thedevice of claim 34 or 36, wherein said thrombin is selected from thegroup consisting of recombinantly produced thrombin and transgenicthrombin.
 38. The device of claim 2, wherein said applicator furthercomprises a sheath or barrel.